Tricyclic Lactams for Use in the Protection of Hematopoietic Stem and Progenitor Cells Against Ionizing Radiation

ABSTRACT

This invention is in the area of tricyclic lactam compounds and methods for protecting healthy cells, and in particular hematopoietic stem and progenitor cells (HSPC), from the damage associated with ionizing radiation (IR) exposure using selective radioprotectants.

RELATED APPLICATIONS

This application claims the benefit of provisional U.S. Application No.61/980,883, filed Apr. 17, 2014, provisional U.S. Application No.61/980,895, filed Apr. 17, 2014, provisional U.S. Application No.61/980,918, filed Apr. 17, 2014, and provisional U.S. Application No.61/980,939, filed Apr. 17, 2014, which are hereby incorporated byreference for all purposes.

GOVERNMENT INTEREST

The U.S. Government has certain rights in this invention arising fromsupport under Grant No. 5R44AI084284 awarded by the National Instituteof Allergy and Infectious Diseases.

FIELD OF THE INVENTION

This invention is in the area of compounds and methods for protectinghealthy cells, and in particular hematopoietic stem and progenitor cells(HSPC), from the damage associated with ionizing radiation (IR) exposureusing selective radioprotectants.

BACKGROUND

Ionizing radiation (IR) is an important therapeutic modality to treat arange of cancers and other proliferative disorders such as tumors.Radiation therapy uses high energy radiation to shrink tumors and killthe proliferating cells. X-rays, gamma rays, and charged particles aretypical kinds of ionizing radiation used for cancer treatments. IRcauses extensive DNA damage to exposed cells, including both normalcells and abnormally proliferating cells such as cancer and tumor cells.

Therapeutic radiation is generally applied to a defined area of thesubject's body which contains abnormal proliferative tissue, in order tominimize the dose absorbed by the nearby normal tissue. It is difficult,however, to selectively administer therapeutic ionizing radiation to theabnormal tissue. Thus, normal tissue proximate to the abnormal tissue isalso exposed to potentially damaging doses of ionizing radiationthroughout the course of treatment. There are also some treatments thatrequire exposure of the subject's entire body to the radiation, in aprocedure called “total body irradiation” (TBI).

Numerous methods have been designed to reduce normal tissue damage whilestill delivering effective therapeutic doses of ionizing radiation.These techniques include brachytherapy, fractionated andhyper-fractionated dosing, complicated dosing scheduling and deliverysystems, and high voltage therapy with a linear accelerator. Suchtechniques, however, only attempt to strike a balance between thetherapeutic and undesirable effects of the radiation and full efficacyhas not been achieved.

In addition, exposure to IR may occur through occupational,environmental, or disaster or terroristic events. For example,occupational doses of ionizing radiation can be received by personswhose job involves exposure to radiation, for example in the nuclearpower and nuclear weapons industry. Incidents such as the 1979 accidentat Three Mile Island or 2011 accident at the Fukushima nuclear powerplant, both of which released radioactive material into the reactorcontainment building and surrounding environment, illustrate thepotential for harmful exposure. Intentional infliction of harmfulradiation can occur during war and aggression.

Hematologic toxicity (i.e., IR-induced bone marrow suppression),resulting in myelosuppression, can be a limiting side-effect associatedwith radiation therapy treatments, resulting in a stoppage, delay, orreduction of treatment until the side-effects subside. Furthermore,hematological toxicity is a major source of morbidity following acuteexposure to high doses of radiation. In particular, proliferatinghematopoietic stem cells and progenitor cells (HSPCs) within the bonemarrow are particularly sensitive to IR, and IR damage to these cellsreduces their ability to reconstitute the hematological cell lineages.For example, exposure to high levels of IR such as total bodyirradiation (TBI) is associated with acute and chronic myelosuppressivehematological toxicities, such as anemia, neutropenia, thrombocytopenia,and lymphcytopenia.

The cytotoxicity of IR, however, is largely cell cycle dependent. Inhealthy cells, cell division occurs in the context of a highly regulatedconcert of molecular events known as the cell cycle. The cell cycle isdivided into four distinct phases: DNA synthesis (S phase), mitosis (Mphase), and the gaps of varying length between these periods called G1and G2. Non-dividing cells remain in a resting or quiescence stage namedG0 before they re-enter into phase G1. Early G1 and late S phases arerelatively radioresistant. Conversely, the G1/S transition and G2/Mphases are relatively radiosensitive (see Sinclair W K, Morton R A.X-ray sensitivity during cell generation cycle of cultured Chinesehamster cells. Radiat. Res. 1966; 29(3):450-474; Terasuna T, Tolmach LJ. X-ray sensitivity and DNA synthesis in synchronous populations ofHeLa cells. Science, 1963; 140:490-92.). Transversing from G1 to S phasewhile harboring DNA damage is particularly toxic. As a result of DNAdamage induced by IR, persistent proliferation in the setting ofunrepaired DNA damage can be fatal to replicating cells (Little J B.Repair of sub-lethal and potentially lethal radiation damage in plateauphase cultures of human cells. Nature, 1969; 224(5221):804-806.). It hasbeen shown that an extended period of G1 after exposure to DNA-damagingagents enhances resistance to such agents, possibly by allowing forgreater DNA repair prior to G1/S transversal (Elkind M M, Sutton H.X-ray damage and recovery in mammalian cells in culture. Nature, 1959;184: 1293-1295; Elkind M M, Sutton H. Radiation response of mammaliancells grown in culture. 1. Repair of x-ray damage in surviving Chinesehamster cells. Radiat Res. 1960; 13: 556-593). Cell cycle arrest allowscells to properly repair these defects, thus preventing theirtransmission to the resulting daughter cells. If repair is unsuccessfulowing to excessive DNA damage, cells may enter senescence or undergoapoptosis.

Hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood as shown in FIG.1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes).HSPCs require the activity of CDK4/6 for proliferation (see Roberts etal. Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in CancerTherapy. JNCI 2012; 104(6):476-487). Hematopoietic cells, however,display a gradient dependency on CDK4/6 activity for proliferationduring myeloid/erythroid differentiation (see Johnson et al. Mitigationof hematological radiation toxicity in mice through pharmacologicalquiescence induced by CDK4/6 inhibition. J Clin. Invest. 2010; 120(7):2528-2536). Accordingly, the least differentiated cells (e.g.,hematopoietic stem cells (HSCs), multi-potent progenitors (MPPs), andcommon myeloid progenitors (CMP)) appear to be the most dependent onCDK4/6 activity for proliferation. More differentiated lineages (e.g.,granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroidprogenitors (MEPs)) are less dependent, and even more differentiatedmyeloid and erythroid cells proliferate independently of CDK4/6activity.

A number of CDK 4/6 inhibitors have been identified, including specificpyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas,benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, andoxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer DrugTargets 8 (2008) 53-75). For example, WO 03/062236 identifies a seriesof 2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment ofRb positive cancers that show selectivity for CDK4/6, including6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991), which is currently being tested by Pfizer/Onyx in clinicaltrials as an anti-neoplastic agent against estrogen-positive,HER2-negative breast cancer. The clinical trial studies have reportedrates of Grade 3/4 neutropenia and leukopenia with the use of PD0332991,resulting in 71% of patients requiring a dose interruption and 35%requiring a dose reduction; and adverse events leading to 10% of thediscontinuations (see Finn, Abstract S1-6, SABCS 2012).

VanderWel et al. describe an iodine-containingpyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387).

WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase andserine/threonine kinase inhibitors.

WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidinecompounds as CDK inhibitors.

WO 2011/101409 also filed by Novartis describes pyrrolopyrimidines withCDK 4/6 inhibitory activity.

WO 2005/052147 filed by Novartis and WO 2006/074985 filed by JanssenPharma disclose additional CDK4 inhibitors.

US 2007/0179118 filed by Barvian et al. teaches the use of CDK4inhibitors to treat inflammation.

U.S. Patent Publication 2011/0224227 to Sharpless et al. describes theuse of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu,et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) toreduce or prevent the effects of cytotoxic compounds on HSPCs in asubject undergoing chemotherapeutic treatments. See also U.S. PatentPublication 2012/0100100.

U.S. Patent Publication 2011/0224221 to Sharpless et al. describes theuse of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu,et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) toreduce or prevent the deleterious effects of ionizing radiation on HSPCsin a subject exposed to radiation. See also U.S. Patent Publication2012/0100100.

Stone, et al., Cancer Research 56, 3199-3202 (Jul. 1, 1996) describesreversible, p16-mediated cell cycle arrest as protection fromchemotherapy.

WO 2012/061156 filed by Tavares and assigned to G1 Therapeuticsdescribes CDK inhibitors (see also, U.S. Pat. Nos. 8,829,012, 8,822,683,8,598,186, 8,691,830, and 8,598,197, all assigned to G1 Therapeutics),describe CDK Inhibitors having the basic core structure:

WO 2013/148748 filed by Tavares and assigned to G1 Therapeuticsdescribes Lactam Kinase inhibitors having the basic core structures:

U.S. Patent Publication 2014/0275066 and 2014/0275067, assigned to G1Therapeutics, describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor the protection of healthy hematopoietic stem and progenitor cells ina subject receiving a DNA-damaging chemotherapeutic agent for thetreatment of a Rb-negative tumors.

U.S. Patent Publication 2014/0274896, assigned to G1 Therapeutics,describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor the protection of healthy hematopoietic stem and progenitor cells ina subject exposed to ionizing radiation.

U.S. Patent Publication 2014/0271466, assigned to G1 Therapeutics,describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor use as an anti-neoplastic for the treatment of a Rb-positiveproliferative disorders.

U.S. Patent Publication 2014/0271460, assigned to G1 Therapeutics,describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor use an anti-neoplastic for the treatment of a T- or B-cell disorder,for example a leukemia.

Stone et al., Cancer Research 56, 3199-3202 (Jul. 1, 1996) describesreversible, p16-mediated cell cycle arrest as protection fromchemotherapy.

Johnson et al. have shown that pharmacological inhibition of CDK4/6using the CDK4/6 inhibitors6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991) and2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4]carbazole-5,6-dione(2BrIC) exhibited IR protective characteristics in CDK4/6-dependent celllines. (Johnson et al. Mitigation of hematological radiation toxicity inmice through pharmacological quiescence induced by CDK4/6 inhibition. JClin. Invest. 2010; 120(7): 2528-2536).

Accordingly, it is an object of the present invention to providecompounds and methods to protect healthy cells, and in particularhematopoietic stem and progenitor cells, during IR exposure.

SUMMARY OF THE INVENTION

Methods and tricyclic lactam compounds are provided to minimize theeffects of ionizing radiation (IR) on hematopoietic stem cells and/orhematopoietic progenitor cells (together referred to as HSPCs) insubjects, typically humans, that will be, are being, or have beenexposed to IR.

Specifically, the invention includes administering an effective amountof a compound of Formula I, II, III, IV, V, or VI, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof, toprotect HSPCs in a subject during or following the subject's exposure toIR. In one non-limiting embodiment, a compound can be selected from thecompounds of Table 1 below, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof.

Compounds of the present invention can be used to protect healthy cellsduring ionizing radiation therapy or radiotherapy for the treatment ofany malignant or non-malignant tumor or abnormal cell proliferation, forexample, in a solid tumor, including a cancer of the brain, breast,cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterus,soft tissue sarcoma, leukemia or lymphoma. The invention can also beused in conjunction with radiotherapy used as a palliative treatment inthe absence of a cure for local control of the tumor or symptomaticrelease, or as a therapeutic treatment to extend the life span of thepatient, or total body irradiation performed prior to bone marrowtransplant. Compounds of the present invention can also be used toprotect healthy cells in connection with radiotherapy for the treatmentof non-malignant conditions, such as trigeminal neuralgia, thyroid eyedisease, pterygium, or prevention of keloid scar growth or heterotopicossification.

The present invention can also be used to protect healthy cells duringionizing radiation therapy or radiotherapy for the treatment ofproliferative disorders, including but not limited to rheumatoidarthritis, lupus, scleroderma, ankylosing spondylitis, asthma,bronchitis and psoriasis. Radiation therapy is also used to treat earlystage Dupuytren's disease and Ledderhose disease.

The present invention can further be used to protect people at imminentrisk of environmental, occupational or aggression-based radiationexposure or who have recently been exposed to harmful radiation.

A compound described herein, in a non-limiting embodiment, may provideprotection of CDK-replication dependent HSPCs during or after IRexposure due in part because it (1) exhibits a transient G1-arrestingeffect and (ii) displays a rapid, synchronous reentry into the cellcycle by the HSPCs following the cessation of IR exposure or mitigationof IR induced DNA damage. The use of CDK4/6-specific transientG1-arresting compounds as radioprotectants and radiomitigants allows foran accelerated hematological recovery, reduced hematologicalcytotoxicity risk due to HSPC replication delay, and/or a minimizationof IR induced cell death.

Tricyclic lactams useful in the present invention can be administered tothe subject prior to exposure to IR, during exposure to IR, afterexposure to IR, or a combination thereof. The compounds described hereinare typically administered in a manner that allows the drug facileaccess to the blood stream, for example via intravenous injection orsublingual, intraaortal, or other efficient blood-stream accessingroute; however, oral or other desired administrative routes can be used.In one embodiment, the compound is administered to the subject less thanabout 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours, 3hours, 2 hours, 1 hour, ½ hour or less prior to exposure to IR. In oneembodiment, the compound is administered up to 4 hours prior to exposureto IR. Typically, the tricyclic lactam is administered to the subjectprior to exposure to IR such that the compound reaches peak serum levelsbefore or during exposure to IR. In one embodiment, the tricyclic lactamis administered concomitantly, or closely thereto, with IR exposure. Inone embodiment, the tricyclic lactam can be administered followingexposure to IR in order to mitigate HSPC DNA damage associated with IRexposure. If desired, the tricyclic lactam can be administered multipletimes during the IR exposure to maximize inhibition, especially when theIR exposure occurs over a long period. In one embodiment, the tricycliclactam is administered up to about ½ hour, up to about 1 hour, up toabout 2 hours, up to about 4 hours, up to about 8 hours, up to about 10hours, up to about 12 hours, up to about 14 hours, up to about 16 hours,up to about 20 hours, or up to about 24 hours or greater following IRexposure. In a particular embodiment, the tricyclic lactam isadministered up to between about 12 hours and 20 hours followingexposure to IR. In one embodiment, the tricyclic lactam is administeredone or more times following exposure to IR.

In one embodiment, the tricyclic lactam compounds inhibit CyclinDependent Kinase 4 (CDK4) and/or Cyclin Dependent Kinase 6 (CDK6). Inone embodiment, the tricyclic lactams useful in the present inventionmay show a marked selectivity for the inhibition of CDK4 and/or CDK6 incomparison to other CDKs, for example CDK2. Tricyclic lactams useful inthe present invention may provide for a dose-dependent G1-arrestingeffect on a subject's HSPCs sufficient to afford radioprotection totargeted HSPCs during IR exposure, while allowing for the reentry intothe cell-cycle by the HSPCs after IR exposure and/or tricyclic lactamadministration due to a time-limited CDK4/6 inhibitory effect. Likewise,tricyclic lactams useful in the present invention may provide adose-dependent mitigating effect on HSPCs that have been exposed to IR,allowing for repair of DNA damage associated with IR exposure.

In addition, in particular embodiments, cell-cycle reentry following G1arrest using a tricyclic lactam described herein may provide for theability to time the administration of hematopoietic growth factors toassist in the reconstitution of hematopoietic cell lines to maximize thegrowth factor effect without forcing hematological cells intoreplication before DNA damage is repaired. As such, in one embodiment,the use of the compounds described herein is combined with the use ofhematopoietic growth factors including, but not limited to, granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steelfactor, and erythropoietin (EPO), and their derivatives. In oneembodiment, the tricyclic lactam is administered prior to administrationof the hematopoietic growth factor. In one embodiment, the hematopoieticgrowth factor administration is timed so that the tricyclic lactam'seffect on HSPCs has dissipated.

In one aspect, the use of a tricyclic lactam described herein allows fora HSPC radio-protective regimen for use during standardradio-therapeutic dosing schedules or regimens common in manyanti-cancer treatments. In some embodiments, the subject is undergoingtherapeutic IR for the treatment of a proliferative disorder or diseasesuch as cancer. In one embodiment, the cancer is a CDK4/6-replicationindependent cancer. In some embodiments, the cancer is characterized byone or more of the group consisting of increased activity ofcyclin-dependent kinase 1 (CDK1), increased activity of cyclin-dependentkinase 2 (CDK2), loss, deficiency, or absence of retinoblastoma tumorsuppressor protein (Rb)(Rb-null), high levels of MYC expression,increased cyclin E, and increased cyclin A. In one embodiment, thesubject is undergoing therapeutic IR for the treatment of an Rb-null orRb-deficient cancer, including but not limited to, small cell lungcancer, triple-negative breast cancer, HPV-positive head and neckcancer, retinoblastoma, Rb-negative bladder cancer, Rb negative prostatecancer, osteosarcoma or cervical cancer. In some cases, administrationof the tricyclic lactam compound allows for a higher dose of ionizingradiation to be used to treat the disease than the standard dose thatwould be safely used in the absence of administration of the tricycliclactam compound.

In some embodiments, the subject is at risk of being exposed to IR dueto an environmental, occupational or aggression-based situation, such asradiological agent exposure during warfare, a radiological terroristattack, an industrial accident, other occupational exposure, or spacetravel.

In some embodiments, the subject has already been exposed to IR, forexample, including but not limited to, through an environmental oroccupational situation, such as radiological agent exposure duringwarfare, a radiological terrorist attack, an industrial accident, otheroccupational exposure, or space travel, and the tricyclic lactamsdescribed herein are administered for the purpose of mitigating DNAdamage in HSPCs.

In some embodiments, the protected HSPCs include hematopoietic stemcells, including long term hematopoietic stem cells (LT-HSCs) and shortterm hematopoietic stem cells (ST-HSCs), and hematopoietic progenitorcells, including multipotent progenitors (MPPs), common myeloidprogenitors (CMPs), common lymphoid progenitors (CLPs),granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroidprogenitors (MEPs). In some embodiments, administration of the tricycliclactam compound provides temporary, transient pharmacologic quiescenceof hematopoietic stem and/or hematopoietic progenitor cells in thesubject.

In one aspect, the methods described herein using a tricyclic lactam mayresult in reduced long-term hematologic toxicity, that is, the use of atricyclic lactam described herein prior to, during, or after IR exposurereduces the occurrence or development of long-term hematologicaltoxicities associated with IR exposure. In some embodiments, thereduction in long-term hematological toxicity is associated with theability of HSPCs that are G1-arrested during IR exposure to thecell-cycle after cessation of IR exposure and replicate, includingreplicating between successive or repeated IR exposures.

Alternatively, administration of a tricyclic lactam as described hereinmay result in reduced anemia, reduced lymphopenia, reducedthrombocytopenia, or reduced neutropenia compared to that typicallyexpected after, common after, or associated with exposure to ionizingradiation in the absence of administration of the tricyclic lactam.

In aspects of the invention, the tricyclic lactam is the compound ofFormula I, II, III, IV, V, or VI. Alternatively, the tricyclic lactamused in the aspects of the invention described herein is selected fromthe compounds of Table 1. In some embodiments, the subject or host is amammal, including a human.

The present invention includes at least the following features:

A. Tricyclic lactam compounds, methods, and compositions for reducingthe effect of IR on CDK4/6 replication dependent healthy cells in asubject, preferably a human, exposed to IR;

B. Tricyclic lactam compounds, methods, and compositions for reducingthe effect of IR on CDK4/6 replication dependent healthy cells, forexample HSPCs, in a subject, preferably a human, undergoing treatmentfor a CDK4/6-replication independent cancer, for example a Rb-null orRb-deficient cancer, comprising administering an effective amount of atricyclic lactam prior to treatment with IR;

C. Tricyclic lactam compounds, methods, and compositions are providedfor reducing the effect of IR exposure on CDK4/6 replication dependentHSPCs in a subject who will be exposed, is being exposed, or has beenexposed to IR, the method comprising administering an effective amountof a tricyclic lactam selected from the group consisting of a compoundor composition comprising Formula I, Formula II, Formula III, FormulaIV, Formula V, and Formula VI, or pharmaceutically acceptablecompositions, salts, isotopic analogs, or prodrugs thereof. In oneembodiment, the compound is selected from the compounds listed in Table1 or pharmaceutically acceptable compositions, salts, isotopic analogs,or prodrugs thereof.

D. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the radioprotection of HSPCs during an IRexposure. In one embodiment, the compound is selected from the compoundslisted in Table 1, or pharmaceutically acceptable compositions, salts,isotopic analogs, or prodrugs thereof;

E. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,or prodrugs thereof, for use in the radioprotection of HSPCs during anIR therapeutic regimen for the treatment of a proliferative disorder. Inone embodiment, the compound is selected from the compounds listed inTable 1, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof;

F. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the radioprotection of HSPCs during an IRtherapeutic regimen for the treatment of cancer. In one embodiment, thecompound is selected from the compounds listed in Table 1, orpharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof;

G. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the radioprotection of HSPCs during an IRtherapeutic regimen for the treatment of a CDK4/6-replicationindependent cancer. In one embodiment, the compound is selected from thecompounds listed in Table 1, or pharmaceutically acceptablecompositions, salts, isotopic analogs, or prodrugs thereof;

H. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the radioprotection of HSPCs during an IRtherapeutic regimen for the treatment of an Rb-null or Rb-deficientcancer. In one embodiment, the compound is selected from the compoundslisted in Table 1, or pharmaceutically acceptable compositions, salts,isotopic analogs, or prodrugs thereof;

I. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the radioprotection of HSPCs during IRexposure associated with an environmental or occupational condition. Inone embodiment, the compound is selected from the compounds listed inTable 1, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof;

J. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,and prodrugs thereof, for use in the forced cycling of HSPCs betweenG1-arrest and replication in coordination with a standard IR therapeuticregimen for a proliferative disorder. In one embodiment, the compound isselected from the compounds listed in Table 1, or pharmaceuticallyacceptable compositions, salts, isotopic analogs, or prodrugs thereof;

K. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the forced cycling of HSPCs betweenG1-arrest and replication in coordination with repeated IR exposures. Inone embodiment, the compound is selected from the compounds listed inTable 1, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof;

L. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in the mitigation of DNA damage to HSPCsfollowing IR exposure. In one embodiment, the compound is selected fromthe compounds listed in Table 1, or pharmaceutically acceptablecompositions, salts, isotopic analogs, or prodrugs thereof;

M. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analogs, orprodrugs thereof, for use in combination with hematopoietic growthfactors in a subject that will be, is being, or has been exposed to IR.In one embodiment, the compound is selected from the compounds listed inTable 1, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof;

N. Use of Compounds of Formula I, II, III, IV, V, and VI as describedherein, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof, in the manufacture of a medicament for usein the radioprotection of HSPCs. In one embodiment, the compound isselected from the compounds listed in Table 1, or pharmaceuticallyacceptable compositions, salts, isotopic analogs, or prodrugs thereof;

O. Use of Compounds of Formula I, II, III, IV, V, and VI as describedherein, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof, in the manufacture of a medicament for usein the mitigation of DNA damage of HSPCs that have been exposed to IR.In one embodiment, the compound is selected from the compounds listed inTable 1, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof;

P. A pharmaceutical formulation comprising an effective subject-treatingamount of compounds of Formula I, II, III, IV, V, and VI as describedherein for the protection against ionizing radiation, orpharmaceutically acceptable compositions, salts, isotopic analog, orprodrugs thereof; In one embodiment, the compound is selected from thecompounds listed in Table 1, or pharmaceutically acceptablecompositions, salts, isotopic analogs, or prodrugs thereof

Q. A method for manufacturing a medicament of Formula I, II, III, IV, V,and VI intended for therapeutic use in the radioprotection of HSPCs. Inone embodiment, the compound is selected from the compounds listed inTable 1, or pharmaceutically acceptable compositions, salts, isotopicanalogs, or prodrugs thereof; and,

R. A method for manufacturing a medicament of Formula I, II, III, IV, V,and VI intended for therapeutic use in the mitigation of DNA damage ofHSPCs that have been exposed to IR. In one embodiment, the medicament isselected from the compounds listed in Table 1, or pharmaceuticallyacceptable compositions, salts, isotopic analogs, or prodrugs thereof.

S. Compounds of Formula I, II, III, IV, V, and VI as described herein,or pharmaceutically acceptable compositions, salts, isotopic analog, orprodrugs thereof; In one embodiment, the compound is selected from thecompounds listed in Table 1, or pharmaceutically acceptablecompositions, salts, isotopic analogs, or prodrugs thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchicalproliferation of healthy hematopoietic stem cells (HSC) and healthyhematopoietic progenitor cells with increasing differentiation uponproliferation.

FIGS. 2-4 illustrate several exemplary embodiments of R² of thecompounds of the invention.

FIGS. 5A-5C, 6A-6D, 7A-7C, 8A-8B and 9A-9F illustrate exemplaryembodiments of the core structure of the compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Tricyclic lactam compounds, methods, and compositions are provided thatminimize the effect of ionizing radiation (IR) toxicity on CDK4/6replication dependent hematopoietic stem cells and/or hematopoieticprogenitor cells (together referred to as HSPCs) in subjects, typicallyhumans, that will be, are being, or have been exposed to IR.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this presently described subject matter belongs. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety to theextent authorized by law.

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise.

Definition of standard chemistry terms may be found in reference works,including Carey and Sundberg (2007) Advanced Organic Chemistry 5^(th)Ed. Vols. A and B, Springer Science+Business Media LLC, New York. Thepractice of the present invention will employ, unless otherwiseindicated, conventional methods of synthetic organic chemistry, massspectroscopy, preparative and analytical methods of chromatography,protein chemistry, biochemistry, recombinant DNA techniques andpharmacology. Conventional methods of organic chemistry include thoseincluded in March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 6^(th) Edition, M. B. Smith and J. March, John Wiley &Sons, Inc., Hoboken, N.J., 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl”and “alkylamino,” embraces linear or branched radicals having one toabout twelve carbon atoms. “Lower alkyl” radicals have one to about sixcarbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl and the like. The term “alkylene” embraces bridging divalentlinear and branched alkyl radicals. Examples include methylene,ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twelve carbon atoms.“Lower alkenyl” radicals having two to about six carbon atoms. Examplesof alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at leastone carbon-carbon triple bond and having two to about twelve carbonatoms. “Lower alkynyl” radicals having two to about six carbon atoms.Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted withone or more functional groups such as halo, hydroxy, nitro, amino,cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino”where amino groups are independently substituted with one alkyl radicaland with two alkyl radicals, respectively. “Lower alkylamino” radicalshave one or two alkyl radicals of one to six carbon atoms attached to anitrogen atom. Suitable alkylamino radicals may be mono or dialkylaminosuch as N-methylamino, N-ethylamino, N.N-dimethylamino, N,N-diethylaminoand the like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of thealkyl carbon atoms is substituted with one or more halo as definedabove. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkylradicals including perhaloalkyl. A monohaloalkyl radical, for oneexample, may have an iodo, bromo, chloro or fluoro atom within theradical. Dihalo and polyhaloalkyl radicals may have two or more of thesame halo atoms or a combination of different halo radicals. “Lowerhaloalkyl” embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Perfluoroalkyl” means an alkyl radical having allhydrogen atoms replaced with fluoro atoms. Examples includetrifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one or two rings wherein such rings may be attachedtogether in a fused manner. The term “aryl” embraces aromatic radicalssuch as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. Morepreferred aryl is phenyl. Said “aryl” group may have 1 or moresubstituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro,cyano, alkoxy, lower alkylamino, and the like. An aryl group may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, andpartially saturated heteroatom-containing ring radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen.Heterocyclic rings comprise monocyclic 6-8 membered rings, as well as5-16 membered bicyclic ring systems (which can include bridged fused andspiro-fused bicyclic ring systems). It does not include rings containing—O—O—.—O—S— or —S—S— portions. Said “heterocyclyl” group may have 1 to 3substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl,lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, and the like.

Examples of saturated heterocyclo groups include saturated 3- to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms[e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,piperazinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,thiazolidinyl]. Examples of partially saturated heterocyclyl radicalsinclude dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl,and the like.

Particular examples of partially saturated and saturated heterocyclogroups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl,thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl,indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl,isochromanyl, chromanyl, 1,2-dihydroquinolyl,1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl,2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl,5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl,3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl,2,3-dihydro-1H-1λ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryland dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicalsare fused/condensed with aryl radicals: unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, for example,indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g.,tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.benzoxazolyl, benzoxadiazolyl]; unsaturated condensed heterocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,benzothiazolyl, benzothiadiazolyl]; and saturated, partially unsaturatedand unsaturated condensed heterocyclic group containing 1 to 2 oxygen orsulfur atoms [e.g. benzofuryl, benzothienyl,2,3-dihydro-benzo[1,4]dioxinyl and dihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized. Examples include unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g.,4H-1,2,4-triazolyl, IH-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated5- to 6-membered heteromonocyclic group containing an oxygen atom, forexample, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, 2-thienyl,3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example,oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

The term “heteroarylalkyl” denotes alkyl radicals substituted with aheteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as“aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula—C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkylradicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examplesinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples includecyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkylradicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicalsattached to alkyl radicals having one to six carbon atoms. Examples ofinclude cyclohexylmethyl. The cycloalkyl in said radicals may beadditionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or morecarbon-carbon double bonds including “cycloalkyldienyl” compounds.Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl andcycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached witha double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which whenadministered to a host in vivo is converted into the parent drug. Asused herein, the term “parent drug” means any of the presently describedchemical compounds that are useful to treat any of the disordersdescribed herein, or to control or improve the underlying cause orsymptoms associated with any physiological or pathological disorderdescribed herein in a host, typically a human. Prodrugs can be used toachieve any desired effect, including to enhance properties of theparent drug or to improve the pharmaceutic or pharmacokinetic propertiesof the parent. Prodrug strategies exist which provide choices inmodulating the conditions for in vivo generation of the parent drug, allof which are deemed included herein. Nonlimiting examples of prodrugstrategies include covalent attachment of removable groups, or removableportions of groups, for example, but not limited to acylation,phosphorylation, phosphonylation, phosphoramidate derivatives,amidation, reduction, oxidation, esterification, alkylation, othercarboxy derivatives, sulfoxy or sulfone derivatives, carbonylation oranhydride, among others.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

The term “selective CDK4/6 inhibitor” and derivatives thereof means acompound that inhibits only CDK4 activity, only CDK6 activity, or bothCDK4 and CDK6 activity at an IC₅₀ molar concentration at least about500, or 1000, or 1500, or 1800, or 2000, or 5000, or 10,000 times lessthan the IC₅₀ molar concentration necessary to inhibit to the samedegree of CDK2 activity in a standard phosphorylation assay.

The term “and/or” when used in describing two items or conditions, e.g.,CDK4 and/or CDK6, refers to situations where both items or conditionsare present or applicable and to situations wherein only one of theitems or conditions is present or applicable. Thus, a CDK4 and/or CDK6inhibitor can be a compound that inhibits both CDK4 and CDK6, a compoundthat inhibits only CDK4, or a compound that only inhibits CDK6.

As described herein, hematopoietic stem and progenitor cells include,but are not limited to, long term hematopoietic stem cells (LT-HSCs),short term hematopoietic stem cells (ST-HSCs), multipotent progenitors(MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors(CLPs), granulocyte-monocyte progenitors (GMPs), andmegakaryocyte-erythroid progenitors (MEPs).

As used herein the term “ionizing radiation” refers to radiation ofsufficient energy that, when absorbed by cells and tissues, can induceformation of reactive oxygen species and DNA damage. Ionizing radiationcan include X-rays, gamma rays, and particle bombardment (e.g., neutronbeam, electron beam, protons, mesons, and others). IR is used forpurposes including, but not limited to, medical testing and treatment,scientific purposes, industrial testing, manufacturing andsterilization, and weapons and weapons development, nuclear energy andcan also be found as an environmental or occupational toxin or used asan assault. Radiation is generally measured in units of absorbed dose,such as the rad or gray (Gy), or in units of dose equivalence, such asrem or sievert (Sv).

By “substantial portion” or “significant portion” is meant at leastabout 80%. In alternative embodiments, the portion may be 85%, 90% or95% or greater.

By “induces G1-arrest” is meant that the tricyclic lactam compoundinduces a quiescent state in a substantial portion of a cell populationat the G1 phase of the cell cycle.

By “long-term hematological toxicity” is meant hematological toxicityaffecting a subject for a period lasting more than one or more weeks,months or years following exposure of IR. Long-term hematologicaltoxicity can result in bone marrow disorders that can cause theineffective production of blood cells (i.e., myelodysplasia) and/orlymphocytes (i.e., lymphopenia, the reduction in the number ofcirculating lymphocytes, such as B- and T-cells). Hematological toxicitycan be observed, for example, as anemia, reduction in platelet count(i.e., thrombocytopenia) or reduction in white blood cell count (i.e.,neutropenia). In some cases, myelodysplasia can result in thedevelopment of leukemia. Long-term toxicity related to ionizingradiation can also damage other self-renewing cells in a subject, inaddition to hematological cells. Thus, long-term toxicity can also leadto graying and frailty.

A tricyclic lactam compound that is “substantially free” of off-targeteffects can have some minor off-target effects that do not interferewith the tricyclic lactam's ability to provide protection from cytotoxiccompounds in CDK4/6-dependent cells. For example, a tricyclic lactamthat is “substantially free” of CDK4/6 inhibitory off-target effects canhave some minor inhibitory effects on other CDKs (e.g., IC₅₀s for CDK1or CDK2 that are >0.5 μM; >1.0 μM, or >5.0 μM), so long as the tricycliclactam provides selective G1 arrest in CDK4/6-dependent cells.

By “synchronous reentry into the cell cycle” is meant that HSPC cells inG1-arrest due to the effects of a tricyclic lactam compound reenter thecell-cycle within relatively the same collective timeframe or atrelatively the same rate upon dissipation of the compound's effect.Comparatively, by “asynchronous reentry into the cell cycle” is meantthat the HSPC cells in G1 arrest reenter the cell-cycle withinrelatively different collective timeframes or at relatively differentrates upon dissipation of the compound's effect, such as induced byPD0332991.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

The subject treated or exposed to IR is typically a human subject,although it is to be understood the methods described herein areeffective with respect to other mammals or vertebrate species. The termsubject can include animals such as mice, monkeys, dogs, pigs, rabbits,domesticated swine (pigs and hogs), ruminants, equine, poultry, felines,murines, bovines, canines, and the like.

Active Compounds

In one embodiment, the invention is directed to compounds or the use ofsuch compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;each X is independently CH or N;each X′ is independently CH or N;X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring;R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl or haloalkyl, cycloalkylor cycloalkyl containing one or more heteroatoms selected from N, O orS; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring;each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0, 1 or 2 and n is 0, 1 or 2;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valence, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring; or R³ and R⁴ together with the nitrogen        atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valence, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valence;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups        may be further independently substituted with one or more        -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,        -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,    -   n is 0, 1 or 2, and    -   m is 0, 1 or 2;        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valence;        or R³* and R⁴* together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valence; and        R⁶ is H or lower alkyl, -(alkylene)m-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valence, and wherein two        R^(x) groups bound to the same or adjacent atoms may optionally        combine to form a ring; and        R¹⁰ is 1 (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        when compounds comprise a double bond in the 6-membered ring        fused to the pyrimidine ring, two R⁸ groups are present and are        as defined above;        when compounds do not comprise a double bond in the 6-membered        ring fused to the pyrimidine ring, four R⁸ groups are present        and are as defined above;        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partially or fully deuterated form thereof

In one embodiment, two R⁸ groups bonded to the same carbon can form anexocyclic double bond. In another embodiment, two R⁸ groups bonded tothe same carbon can form a carbonyl group.

In one embodiment, the invention is directed to compounds or the use ofsuch compounds of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as definedabove;each R¹⁴ is independently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl orcycloalkyl containing one or more heteroatoms selected from N, O or S;-(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring;or two R¹⁴ groups bonded to the same carbon can form an exocyclic doublebond;or two R¹⁴ groups bonded to the same carbon can form a carbonyl group;andwhen the compound of Formula VI has a double bond, as indicated by the(----), in the 6-membered ring fused to the pyrimidine ring, two R¹⁴groups are present as allowed for in Formula VI above; orwhen the compound of Formula VI does not include a double bond, asindicated by the (----), in the 6-membered ring fused to the pyrimidinering, four R¹⁴ groups are present as allowed for in Formula VI above;or a pharmaceutically acceptable salt, prodrug or isotopic variant, forexample, partially or fully deuterated form thereof

In an alternative embodiment, the invention is directed to compounds orthe use of such compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;each X is independently CH or N;each X′ is independently CH or N;X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring;R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl (including methyl) orhaloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatomsselected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring;each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valance, andwherein two R^(x) groups bound to the same or adjacent atom mayoptionally combine to form a ring and wherein m is 0, 1, or 2 and n is0, 1 or 2;wherein heterocyclo may be optionally independently substituted with 1to 3 R^(x) groups as allowed by valance, and wherein two R^(x) groupsbound to the same or adjacent atom may optionally combine to form aring;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which, other than heterocyclo, may be optionally        independently substituted with one or more R^(x) groups as        allowed by valance, and wherein two R^(x) groups bound to the        same or adjacent atom may optionally combine to form a ring; or        R³ and R⁴ together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valance, and wherein two R^(x) groups bound to the        same or adjacent atom may optionally combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which, other than heterocyclo, may be        optionally independently substituted with one or more R^(x)        groups as allowed by valance;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵, -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups,        any of which, other than heterocyclo, may be further        independently substituted with one or more -(alkylene)_(m)-CN,        -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,        -(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,        -(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,        -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,        -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴* ,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴* ,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, and    -   wherein heterocycle may be further independently substituted        with one to three substitutions selected from    -   -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,        -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*;    -   n is 0, 1 or 2, and    -   m is 0, 1; or 2 and        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which, other than heterocyclo, may be        optionally independently substituted with one or more R^(x)        groups as allowed by valance; or R³* and R⁴* together with the        nitrogen atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valance;        R⁶ is H, absent, or lower alkyl, -(alkylene)m-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which, other than heterocyclo, may be optionally        independently substituted with one or more R^(x) groups as        allowed by valence, and wherein two R^(x) groups bound to the        same or adjacent atoms may optionally combine to form a ring;        and        R¹⁰ is 1 (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        when the compound of Formula I, II, III, IV, or V has a double        bond, as indicated by the (----), in the 6-membered ring fused        to the pyrimidine ring, two R⁸ groups are present as allowed for        in Formula I, II, III, IV, or V above; or        when the compound of Formula I, II, III, IV, or V does not        include a double bond, as indicated by the (----), in the        6-membered ring fused to the pyrimidine ring, four R⁸ groups are        present as allowed for in Formula I, II, III, IV, or V above;        wherein each heteroaryl is an aryl ring system that contains one        or more heteroatoms selected from the group O, N and S, wherein        the ring nitrogen and sulfur atom(s) are optionally oxidized,        and nitrogen atom(s) are optionally quarternized;        wherein each aryl is a carbocyclic aromatic system containing        one or two rings, wherein such rings may be attached together in        a fused manner, and wherein each aryl may have 1 or more R^(x)        substituents;        wherein each heterocyclo is a saturated or partially saturated        heteroatom-containing ring radical, where the heteroatoms may be        selected from nitrogen, sulfur and oxygen, wherein each        heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered        bicyclic ring system, and wherein each heterocyclo may have 1 to        3 R^(x) substituents;        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partially or fully deuterated form        thereof.

In an alternative embodiment, the term “aryl” means a carbocyclicaromatic system containing one or two rings wherein such rings may beattached together in a fused manner, which may have 1 or moresubstituents selected from lower alkyl, hydroxyl, halo, haloalkyl,nitro, cyano, alkoxy and lower alkylamino.

In an alternative embodiment, the term “heterocyclyl” or “heterocyclo”means a saturated or partially saturated heteroatom-containing ringradical, where the heteroatoms may be selected from nitrogen, sulfur andoxygen, which may have 1 to 3 substituents selected from hydroxyl, Boc,halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy,amino and lower alkylamino, wherein the heterocyclic ring is amonocyclic 6-8 membered rings, or a 5-16 membered bicyclic ring systemswhich can include bridged fused and spiro-fused bicyclic ring systems,and which does not include rings containing —O—O—.—O—S— or —S—S—portion.

In an alternative embodiment, the term “heteroaryl” means an aryl ringsystem that contains one or more heteroatoms selected from the group O,N and S, wherein the ring nitrogen and sulfur atom(s) are optionallyoxidized, and nitrogen atom(s) are optionally quarternized.

In one embodiment, two R⁸ groups bonded to the same carbon can form anexocyclic double bond. In another embodiment, two R⁸ groups bonded tothe same carbon can form a carbonyl group.

In an alternative embodiment, the invention is directed to compounds orthe use of such compounds of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as definedabove;each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) orhaloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatomsselected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)—NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring;or two R¹⁴ groups bonded to the same carbon can form an exocyclic doublebond;or two R¹⁴ groups bonded to the same carbon can form a carbonyl group;andwhen the compound of Formula VI has a double bond, as indicated by the(----), in the 6-membered ring fused to the pyrimidine ring, two R¹⁴groups are present as allowed for in Formula VI above; orwhen the compound of Formula VI does not include a double bond, asindicated by the (----), in the 6-membered ring fused to the pyrimidinering, four R¹⁴ groups are present as allowed for in Formula VI above;wherein each heteroaryl is an aryl ring system that contains one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized;wherein each aryl is a carbocyclic aromatic system containing one or tworings, wherein such rings may be attached together in a fused manner,and wherein each aryl may have 1 or more R^(x) substituents;wherein each heterocyclo is a saturated or partially saturatedheteroatom-containing ring radical, where the heteroatoms may beselected from nitrogen, sulfur and oxygen, wherein each heterocyclo is amonocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system,and wherein each heterocyclo may have 1 to 3 R^(x) substituents;or a pharmaceutically acceptable salt, prodrug or isotopic variant, forexample, partially or fully deuterated form thereof. In some aspects,the compound is selected from Formula I or Formula II and R⁶ is absent.

In some aspects, the compound is of Formula III:

and the variables are as defined for compounds of Formulae I and II andpharmaceutically acceptable salts, isotopic analogs, or prodrugsthereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may beoptionally independently substituted with one or more R^(x) groups asallowed by valence, and wherein two R^(x) groups bound to the same oradjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without furthersubstitution.

In some aspects, m in R² is 1. In a further aspect, the alkylene in R²is methylene.

In some aspects, R² is

wherein:R²* is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)-, or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;each R^(x1) is independently-(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)wherein each m is independently 0 or 1 provided at least one m is 1,—(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0 or 1,—N(R^(N))-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)_(m)-heterocyclylwherein m is 0 or 1, or —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,—S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:

-   -   R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and    -   wherein two R^(x1) can, together with the atoms to which they        attach on P, which may be the same atom, form a ring; and        t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted byunsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclylwherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:R²* is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)-, or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;P1 is a 4- to 6-membered monocyclic saturated heterocyclyl group;each R^(x2) is independently hydrogen or alkyl; ands is 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is notfurther substituted.

In some aspects, R² is selected from the structures depicted in FIGS.2-4.

In some aspects, R² is

In some aspects, the compound has general Formula I and morespecifically one of the general structures in FIGS. 5A-9F wherein thevariables are as previously defined.

In some aspects, the compound has general Formula Ia:

wherein R¹, R², R, R⁸, X and y are as previously defined.

In some embodiments, the compound has Formula Ia and R is alkyl.

In some embodiments, the compound has Formula Ia and R is H.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl andR²* is as previously defined.

In some embodiments, the compound is of Formula Ib:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound is of Formula Ib and R is alkyl.

In some embodiments, the compound is of Formula Ib and R is H.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ic:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ic and R is alkyl.

In some embodiments, the compound has Formula Ic and R is H.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Id:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Id and R is alkyl.

In some embodiments, the compound has Formula Id and R is H.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ie:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ie and R is alkyl.

In some embodiments, the compound has Formula Ie and R is H.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula If:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula If and R is alkyl.

In some embodiments, the compound has Formula If and R is H.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ig:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ig and R is alkyl.

In some embodiments, the compound has Formula Ig and R is H.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ih:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ih and R is alkyl.

In some embodiments, the compound has Formula Ih and R is H.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ii:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ii and R is alkyl.

In some embodiments, the compound has Formula Ii and R is H.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ij:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ij and R is alkyl.

In some embodiments, the compound has Formula Ij and R is H.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Ij and R is H, and X is CHand N.

In some embodiments, the compound has the structure:

In some embodiments, the compound has the structure Ik:

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Il:

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Im:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIa:

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIb:

In some embodiments, the compound has Formula IIb and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula IIb and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the active compound is:

Further specific compounds that fall within the present invention andthat can be used in the disclosed methods of treatment and compositionsinclude, but are not limited to, the structures listed in Table 1 below.

TABLE 1 Structures of Tricyclic Lactams Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX

YYY

ZZZ

AAAA

BBBB

CCCC

DDDD

EEEE

FFFF

GGGG

HHHH

Isotopic Substitution

The present invention includes compounds and the use of compounds withdesired isotopic substitutions of atoms, at amounts above the naturalabundance of the isotope, i.e., enriched. Isotopes are atoms having thesame atomic number but different mass numbers, i.e., the same number ofprotons but a different number of neutrons. By way of general exampleand without limitation, isotopes of hydrogen, for example, deuterium(²H) and tritium (³H) may be used anywhere in described structures.Alternatively or in addition, isotopes of carbon, e.g., 13C and ¹⁴C, maybe used. A preferred isotopic substitution is deuterium for hydrogen atone or more locations on the molecule to improve the performance of thedrug. The deuterium can be bound in a location of bond breakage duringmetabolism (an α-deuterium kinetic isotope effect) or next to or nearthe site of bond breakage (a β-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise. The term “isotopically-labeled” analogrefers to an analog that is a “deuterated analog”, a “¹³C-labeledanalog,” or a “deuterated/¹³C-labeled analog.” The term “deuteratedanalog” means a compound described herein, whereby a H-isotope, i.e.,hydrogen/protium (¹H), is substituted by a H-isotope, i.e., deuterium(²H). Deuterium substitution can be partial or complete. Partialdeuterium substitution means that at least one hydrogen is substitutedby at least one deuterium. In certain embodiments, the isotope is 90, 95or 99% or more enriched in an isotope at any location of interest. Insome embodiments it is deuterium that is 90, 95 or 99% enriched at adesired location.

Hematopoietic Stem Cells and Cyclin-Dependent Kinase Inhibitors

Tissue-specific stem cells are capable of self-renewal, meaning thatthey are capable of replacing themselves throughout the adult mammalianlifespan through regulated replication. Additionally, stem cells divideasymmetrically to produce “progeny” or “progenitor” cells that in turnproduce various components of a given organ. For example, in thehematopoietic system, the hematopoietic stem cells give rise toprogenitor cells which in turn give rise to all the differentiatedcomponents of blood (e.g., white blood cells, red blood cells, andplatelets) (See FIG. 1).

Early hematopoietic stem/progenitor cells (HSPC) in the adult mammalrequire the enzymatic activity of the proliferative kinasescyclin-dependent kinase 4 (CDK4) and/or cyclin-dependent kinase 6 (CDK6)for cellular replication. In contrast, the majority of proliferatingcells in adult mammals (e.g., the more differentiated blood-formingcells in the bone marrow) do not require the activity of CDK4 and/orCDK6 (i.e., CDK4/6). These differentiated cells can proliferate in theabsence of CDK4/6 activity by using other proliferative kinases, such ascyclin-dependent kinase 2 (CDK2) or cyclin-dependent kinase 1 (CDK1).

The present invention includes methods of protecting healthy cells in asubject, and in particular, hematopoietic cells and/or progenitor cells(HSPCs) from the toxic effects or mitigation of ionizing radiation bythe administration of a tricyclic lactam compound. In one embodiment,the tricyclic lactam compound is a selective CDK4/6 inhibitor. The useof tricyclic lactams as CDK4/6-specific G1-arresting effect compounds asradioprotectants and radiomitigants allows for an acceleratedhematological recovery and reduced hematological cytotoxicity risk dueto HSPC replication delay. In certain embodiments, the tricyclic lactamadministered is selected from the group consisting of a compound orcomposition comprising Formula I, Formula II, Formula III, Formula IV,Formula V, Formula VI, or a combination thereof. In one non-limitingembodiment, a compound can be selected from the compounds of Table 1, ora pharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof.

In certain aspects, compounds, methods, and compositions are providedfor reducing or limiting the effect of DNA damaging ionizing radiationon hematopoietic stem and progenitor cells in a subject undergoingtreatment for a Rb-null cancer, the method comprising administering aneffective amount of a tricyclic compound prior to exposure to IR. In oneembodiment, a substantial portion of the hematopoietic stem and/orprogenitor cells return to pre-treatment baseline cell cycle activity(i.e., reenter the cell-cycle) within less than about 48 hours ofadministration of the tricyclic lactam. In certain embodiments, thetricyclic lactam administered is selected from the group consisting of acompound or composition comprising Formula I, Formula II, Formula III,Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof. In onenon-limiting embodiment, a compound can be selected from the compoundsof Table 1, or a pharmaceutically acceptable composition, salt, isotopicanalog, or prodrug thereof.

In certain aspects, tricyclic lactam compounds, methods, and compositionare provided for reducing or limiting the effect of DNA-damaging IR onhematopoietic stem and progenitor cells in a subject that has beenexposed to IR, the method comprising administering an effective amountof a tricyclic lactam following exposure to IR, wherein a substantialportion of the hematopoietic stem and/or progenitor cells reenter thecell-cycle synchronously within less than about 24, 30, 36, 40, or 48hours following the dissipation of the compound's CDK4/6 inhibitoryeffect, wherein the tricyclic lactam compound has an IC₅₀ CDK4inhibitory concentration that is more than 500 times less than its IC₅₀inhibitory concentration for CDK2. In certain embodiments, a substantialportion of the hematopoietic stem and/or progenitor cells reenter thecell-cycle synchronously within less than about 24, 30, 36, 40, or 48hours from the point in which the tricyclic lactam's concentration levelin the subject's blood drops below a therapeutic effectiveconcentration. In certain embodiments, the tricyclic lactam administeredis selected from the group consisting of a compound or compositioncomprising Formula I, Formula II, Formula III, Formula IV, Formula V,and Formula VI or a pharmaceutically acceptable composition, salt,isotopic analog, or prodrug thereof. In one non-limiting embodiment, acompound can be selected from the compounds of Table 1, or apharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof.

In certain embodiments, the tricyclic lactam is a CDK4/6 inhibitorselected from Formula I, II, III, IV, V, or VI, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof,wherein the protection afforded by the compound is short term andtransient in nature, allowing a significant portion of the cells tosynchronously renter the cell-cycle following the cessation of IRexposure. Cells that are quiescent within the G1 phase of the cell cycleare more resistant to the DNA damaging effect of radiation thanproliferating cells.

In one embodiment, the tricyclic lactam compounds for use in thedescribed methods are CDK4/6 inhibitors, with minimal CDK2 inhibitoryactivity. In one embodiment, a tricyclic lactam compound for use in themethods described herein has a CDK4/CycD1 IC₅₀ inhibitory concentrationvalue thatis >100, >200, >300, >400, >500, >600, >700, >800, >900, >1000, >1250, >1500times, >1800 times, >2000 times, >2200 times, >2500 times, >2700times, >3000 times, >3200 times lower than its respective IC₅₀concentration value for CDK2/CycE inhibition. In one embodiment, atricyclic lactam for use in the methods described herein has an IC₅₀concentration value for CDK4/CycD1 inhibition that is about <1.50 nM,<1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM, <0.70 nM,<0.65 nM, <0.60 nM, <0.55 nM, or less. In one embodiment, a tricycliclactam for use in the methods described herein has an IC₅₀ concentrationvalue for CDK2/CycE inhibition that is about >1.0 μM, >1.25 μM, >1.50μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75 μM, >3.0 μM, >3.25μM, >3.5 μM or greater. In one embodiment, a tricyclic lactam for use inthe methods described herein has an IC₅₀ concentration value forCDK2/CycA IC₅₀ that is >0.80 μM, >0.85 μM, >0.90 μM, >0.95 μM, >0.1.0μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75μM, >3.0 μM or greater. In one embodiment, the tricyclic lactam for usein the methods described herein are selected from the group consistingof Formula I, Formula II, Formula III, Formula IV, Formula V, andFormula VI, or a pharmaceutically acceptable composition, salt, isotopicanalog, or prodrug, thereof. In one non-limiting embodiment, a compoundcan be selected from the compounds of Table 1, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof.

According to the presently disclosed subject matter, radiationprotection with the tricyclic lactams described herein may be achievedby a number of different dosing schedules. In addition to multi-dosingschedules or single pretreatment, concomitant treatment can also beeffective.

In one embodiment, the tricyclic lactams described herein are used inHSPC cycling strategies wherein a subject is exposed to regular,repeated IR exposures, wherein HSPCs are G1-arrested when IR exposed andallowed to reenter the cell-cycle before the subject's next IR exposure.Such cycling allows HSPCs to regenerate damaged blood cell lineages inbetween regular, repeated IR exposures, for example those associatedwith standard IR treatments for cancer, and reduces the risk associatedwith long term CDK4/6 inhibition.

In one embodiment, the subject is exposed to IR at least 5 times a week,at least 4 times a week, at least 3 times a week, at least 2 times aweek, at least 1 time a week, at least 3 times a month, at least 2 timesa month, or at least 1 time a month, wherein the subject's HSPCs are G1arrested during treatment and allowed to cycle in between IR exposure,for example during a treatment break. In one embodiment, the subject isundergoing 5 times a week IR exposure, wherein the subject's HSPCs areG1 arrested during the IR exposure and allowed to reenter the cell-cycleduring the 2 day break, for example, over the weekend.

In one embodiment, using a tricyclic lactam described herein, thesubject's HSPCs are arrested during the entirety of the IR exposuretime-period for the weekly treatment, for example, during a 5 times/weekIR regimen, the cells are arrested over the time period that is requiredto complete the IR exposure regimen for the week, and then allowed torecycle at the end of the regimen. In one embodiment, using a tricycliclactam described herein, the subject's HSPCs are arrested during theentirety of the IR regimen, for example, in a 5 times a week IR regimenfor 5 weeks, and rapidly reenter the cell-cycle following the completionof the IR regimen.

In one embodiment, the subject has been exposed to IR, and, using atricyclic lactam described herein, the subject's HSPCs are placed in G1arrest following exposure in order to mitigate DNA damage. In oneembodiment, the tricyclic lactam is administered at least ½ hour, atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, atleast 10 hours, at least 12 hours, at least 14 hours, at least 16 hours,at least 18 hours, at least 20 hours, at least 24 hours or more post IRexposure. In one embodiment, the subject has been exposed to IR and isadministered multiple tricyclic lactam doses at differing time points,for example, at 12 hours and 24 hours post IR exposure.

In some embodiments, the presently disclosed subject matter providesmethods for protection of mammals from the acute and chronic toxiceffects of ionizing radiation by forcing hematopoietic stem andprogenitor cells (HSPCs) into a quiescent state by transient (e.g., overa period of less than about 40 hours, 36 hours, 30 hours, 24 hours, 20hours, 16 hours, 12 hours, 8 hours, 4 hours, 3 hours, 2.5 hours, 2hours, 1 hour, ½ hour or less prior to IR exposure) treatment with atricyclic lactam selected from the group consisting of Formula I,Formula II, Formula III, Formula IV, Formula V, and Formula VI, or apharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. HSPCs recover from this period of transient quiescence,and then function normally after treatment with the compound is stopped,and its intra-cellular effect dissipates. During the period ofquiescence, the stem and progenitor cells are protected from the effectsof ionizing radiation. The ability to protect stem/progenitor cells isdesirable both in the treatment of cancer where patients are given high,repeated doses of ionizing radiation, and in environmental oroccupational situations where individuals may be in danger of beingexposed to large doses of radiation. In some embodiments, the HSPCs canbe arrested for longer periods, for example, over a period of hours,days, and/or weeks, through multiple, time separated administrations ofa tricyclic lactam described herein.

In one embodiment of the invention, these compounds can be administeredin a concerted regimen with a blood growth factor agent. As such, in oneembodiment, the use of the compounds and methods described herein iscombined with the use of hematopoietic growth factors including, but notlimited to, granulocyte colony stimulating factor (G-CSF, for example,sold as Neupogen (filgrastin), Neulasta (peg-filgrastin), orlenograstin), granulocyte-macrophage colony stimulating factor (GM-CSF,for example sold as molgramostim and sargramostim (Leukine)), M-CSF(macrophage colony stimulating factor), thrombopoietin (megakaryocytegrowth development factor (MGDF), for example sold as Romiplostim andEltrombopag) interleukin (IL)-12, interleukin-3, interleukin-11(adipogenesis inhibiting factor or oprelvekin), SCF (stem cell factor,steel factor, kit-ligand, or KL) and erythropoietin (EPO), and theirderivatives (sold as for example epoetin-α as Darbopoetin, Epocept,Nanokine, Epofit, Epogin, Eprex and Procrit; epoetin-β sold as forexample NeoRecormon, Recormon and Micera), epoetin-delta (sold as forexample Dynepo), epoetin omega (sold as for example Epomax), epoetinzeta (sold as for example Silapo and Reacrit) as well as for exampleEpocept, EPOTrust, Erypro Safe, Repoeitin, Vintor, Epofit, Erykine,Wepox, Espogen, Relipoeitin, Shanpoietin, Zyrop and EPIAO).

It has been recently reported that some of the hematopoietic growthfactors can have serious side effects. For example, the EPO family oftherapeutics has been associated with arterial hypertension, cerebralconvulsions, hypertensive encephalopathy, tumor progressionthromboembolism, iron deficiency, influenza like syndromes and venousthrombosis. The G-CSF family of therapeutics has been associated withmyelodysplasia and secondary leukemia, spleen enlargement and rupture,respiratory distress syndrome, allergic reactions and sickle cellcomplications.

By combining the administration of the described tricyclic lactams andmethods of the present invention with hematopoietic growth factors, itis possible for the health care practitioner to decrease the amount ofthe growth factor to minimize the unwanted adverse effects whileachieving the therapeutic benefit. Thus, in this embodiment, thetricyclic lactam allows the patient to receive some amount of the growthfactor. The patient may not need as much hematopoietic growth factorbecause the hematopoietic cells will have been protected during thechemotherapy and not diminished to the extent without the tricycliclactam. Furthermore, by timing the administration of the growth factors,hematopoietic cells are not forced into replicating while harboringmajor DNA structural damage.

Several advantages can result from the radio-protective methodsdescribed herein using a tricyclic lactam described herein. Thereduction in radio-toxicity afforded by the tricyclic lactam can allowfor dose intensification (e.g., more therapy can be given in a fixedperiod of time) in medically related IR therapies, which will translateto better efficacy. Therefore, the presently disclosed methods canresult in radio-therapy regimens that are less toxic and more effective.Also, in contrast to protective treatments with exogenous biologicalgrowth factors, in one embodiment, the tricyclic lactam described hereinare orally available small molecules, which can be formulated foradministration via a number of different routes. When appropriate, suchsmall molecules can be formulated for oral, topical, intranasal,inhalation, intravenous, intramuscular, or any other form ofadministration. Further, as opposed to biologics, stable small moleculescan be more easily stockpiled and stored. Thus, the tricyclic lactamcompounds can be more easily and cheaply kept on hand in emergency roomswhere subjects of IR exposure can report or at sites where radiationexposure is particularly likely to occur: at nuclear power plants, onnuclear powered vessels, at military installations, near battlefields,etc.

In one embodiment, the use of a tricyclic lactam as described herein caninduce selective G1 arrest in CDK4/6-dependent cells (e.g., as measuredin a cell-based in vitro assay). In one embodiment, the tricyclic lactamis capable of increasing the percentage of CDK4/6-dependent cells in theG1 phase, while decreasing the percentage of CDK4/6-dependent cells inthe G2/M phase and S phase. In one embodiment, the tricyclic lactaminduces substantially pure (i.e., “clean”) G1 cell cycle arrest in theCDK4/6-dependent cells (e.g., wherein treatment with the tricycliclactam induces cell cycle arrest such that the majority of cells arearrested in G1 as defined by standard methods (e.g. propidium iodide(PI) staining or others) with the population of cells in the G2/M and Sphases combined being less than about 30%, about 25%, about 20%, about15%, about 10%, about 5%, about 3% or less of the total cell population.Methods of assessing the cell phase of a population of cells are knownin the art (see, for example, in U.S. Patent Application Publication No.2002/0224522) and include cytometric analysis, microscopic analysis,gradient centrifugation, elutriation, fluorescence techniques includingimmunofluorescence, and combinations thereof. Cytometric techniquesinclude exposing the cell to a labeling agent or stain, such asDNA-binding dyes, e.g., PI, and analyzing cellular DNA content by flowcytometry. Immunofluorescence techniques include detection of specificcell cycle indicators such as, for example, thymidine analogs (e.g.,5-bromo-2-deoxyuridine (BrdU) or an iododeoxyuridine), with fluorescentantibodies.

In some embodiments, the use of a tricyclic lactam described hereinreduces the risk of undesirable off-target effects including, but notlimited to, long term toxicity, anti-oxidant effects, and estrogeniceffects. Anti-oxidant effects can be determined by standard assays knownin the art. For example, a compound with no significant anti-oxidanteffects is a compound that does not significantly scavengefree-radicals, such as oxygen radicals. The anti-oxidant effects of acompound can be compared to a compound with known anti-oxidant activity,such as genistein. Thus, a compound with no significant anti-oxidantactivity can be one that has less than about 2, 3, 5, 10, 30, or 100fold anti-oxidant activity relative to genistein. Estrogenic activitiescan also be determined via known assays. For instance, a non-estrogeniccompound is one that does not significantly bind and activate theestrogen receptor. A compound that is substantially free of estrogeniceffects can be one that has less than about 2, 3, 5, 10, 20, or 100 foldestrogenic activity relative to a compound with estrogenic activity,e.g., genistein.

In some embodiments, the subject has been exposed to ionizing radiation,will be exposed to ionizing radiation, or is at risk of incurringexposure to ionizing radiation as the result of radiological agentexposure during warfare, a radiological terrorist attack, an industrialaccident, or space travel. Subjects can further be exposed to, or bescheduled to be exposed to, ionizing radiation when undergoingtherapeutic irradiation for the treatment of proliferative disorders.Such disorders include cancerous and non-cancer proliferative diseases.The compounds are effective in protecting healthy hematopoieticstem/progenitor cells during therapeutic irradiation of a broad range oftumor types, including but not limited to the following: breast,prostate, ovarian, skin, lung, colorectal, brain (i.e., glioma) andrenal. Ideally, growth of the cancer being treated by IR should not beaffected by the tricyclic lactam compound. The potential sensitivity ofcertain tumors to CDK4/6 inhibition can be deduced based on tumor typeand molecular genetics using standard techniques. Cancers that are nottypically affected by the inhibition of CDK4/6 are those that can becharacterized by one or more of the group including, but not limited to,increased activity of CDK1 or CDK2, loss or absence of retinoblastoma(Rb) tumor suppressor protein (Rb-null), high levels of MYC expression,increased cyclin E and increased cyclin A. Such cancers can include, butare not limited to, small cell lung cancer, retinoblastoma, HPV positivemalignancies like cervical cancer and certain head and neck cancers, MYCamplified tumors such as certain classes of Rb-positive BurkittsLymphoma, and triple negative breast cancer; certain classes of sarcoma,certain classes of non-small cell lung carcinoma, certain classes ofmelanoma, certain classes of pancreatic cancer, certain classes ofleukemias, certain classes of lymphomas, certain classes of braincancer, certain classes of colon cancer, certain classes of prostatecancer, certain classes of ovarian cancer, certain classes of uterinecancer, certain classes of thyroid and other endocrine tissue cancers,certain classes of salivary cancers, certain classes of thymiccarcinomas, certain classes of kidney cancers, certain classes ofbladder cancer and certain classes of testicular cancers.

The loss or absence of retinoblastoma (Rb) tumor suppressor protein(Rb-null) can be determined through any of the standard assays known toone of ordinary skill in the art, including but not limited to WesternBlot, ELISA (enzyme linked immunoadsorbent assay), IHC(immunohistochemistry), and FACS (fluorescent activated cell sorting).The selection of the assay will depend upon the tissue, cell line orsurrogate tissue sample that is utilized e.g., for example Western Blotand ELISA may be used with any or all types of tissues, cell lines orsurrogate tissues, whereas the IHC method would be more appropriatewherein the tissue utilized in the methods of the present invention wasa tumor biopsy. FACs analysis would be most applicable to samples thatwere single cell suspensions such as cell lines and isolated peripheralblood mononuclear cells. See for example, US 20070212736 “FunctionalImmunohistochemical Cell Cycle Analysis as a Prognostic Indicator forCancer”.

Alternatively, molecular genetic testing may be used for determinationof retinoblastoma gene status. Molecular genetic testing forretinoblastoma includes the following as described in Lohmann and Gallie“Retinoblastoma. Gene Reviews” (2010)http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastomaor Parsam et al. “A comprehensive, sensitive and economical approach forthe detection of mutations in the RB 1 gene in retinoblastoma” Journalof Genetics, 88(4), 517-527 (2009).

Increased activity of CDK1 or CDK2, high levels of MYC expression,increased cyclin E and increased cyclin A can be determined through anyof the standard assays known to one of ordinary skill in the art,including but not limited to Western Blot, ELISA (enzyme linkedimmunoadsorbent assay), IHC (immunohistochemistry), and FACS(fluorescent activated cell sorting). The selection of the assay willdepend upon the tissue, cell line or surrogate tissue sample that isutilized e.g., for example Western Blot and ELISA may be used with anyor all types of tissues, cell lines or surrogate tissues, whereas theIHC method would be more appropriate wherein the tissue utilized in themethods of the present invention was a tumor biopsy. FACs analysis wouldbe most applicable to samples that were single cell suspensions such ascell lines and isolated peripheral blood mononuclear cells.

In some embodiments, the cancer a small cell lung cancer,retinoblastoma, and triple negative (ER/PR/Her2 negative) or“basal-like” breast cancer, which almost always inactivate theretinoblastoma tumor suppressor protein (Rb), and therefore do notrequire CDK4/6 activity to proliferate. Triple negative (basal-like)breast cancer is also almost always genetically or functionally Rb-null.Also, certain virally induced cancers (e.g. cervical cancer and subsetsof Head and Neck cancer) express a viral protein (E7) which inactivatesRb making these tumors functionally Rb-null. Some lung cancers are alsobelieved to be caused by HPV.

The tricyclic lactams described herein can also be used in protectinghealthy CDK4/6-replication dependent cells during ionizing radiation ofabnormal tissues in non-cancer proliferative diseases, including but notlimited to the following: psoriasis, lupus, arthritis (notablyrheumatoid arthritis), hemangiomatosis in infants, multiple sclerosis,myelodegenerative disease, neurofibromatosis, ganglioneuromatosis,keloid formation, Paget's Disease of the bone, fibrocystic disease ofthe breast, Peyronie's and Duputren's fibrosis, restenosis, andcirrhosis.

According to the present invention, therapeutic ionizing radiation canbe administered to a subject on any schedule and in any dose consistentwith the prescribed course of treatment, for example by administering acompound of Formula I, Formula II, Formula III, Formula IV, Formula V,and Formula VI, or a compound selected from Table 1, prior to or duringthe radiation. Preferably, administration of the compound is timed suchthat maximal G1 arrest of the HSPCs, or a significant portion thereof,occurs at the time of the IR exposure. In certain embodiments, atricyclic lactam compound described herein is administered so that apeak serum concentration for the compound is reached at or near the timeof IR exposure. If desired, multiple doses of the radioprotectantcompound can be administered to the subject. Alternatively, the subjectcan be given a single dose of the compound. The course of treatmentdiffers from subject to subject, and those of ordinary skill in the artcan readily determine the appropriate dose and schedule of therapeuticradiation in a given clinical situation.

Active Compounds, Salts and Formulations

As used herein, the term “active compound” refers to the tricycliclactam compounds described herein or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof. The activecompound can be administered to the subject through any suitableapproach. The amount and timing of active compound administered isdependent on the subject being treated, on the dosage of IR to which thesubject is anticipated of being exposed to, on the time course of the IRexposure, on the manner of administration, on the pharmacokineticproperties of the particular active compound, and on the judgment of theprescribing physician. Thus, because of subject to subject variability,the dosages given below are a guideline and the physician can titratedoses of the compound to achieve the treatment that the physicianconsiders appropriate for the subject. In considering the degree oftreatment desired, the physician can balance a variety of factors suchas age and weight of the subject, presence of preexisting disease, aswell as presence of other diseases. Pharmaceutical formulations can beprepared for any desired route of administration including, but notlimited to, oral, intravenous, or aerosol administration, as discussedin greater detail below.

The therapeutically effective dosage of any of the active compounddescribed herein will be determined by the health care practitionerdepending on the condition, size and age of the patient as well as theroute of delivery. In one embodiment, a dosage from about 0.1 to about200 mg/kg is administered, with all weights being calculated based uponthe weight of the active compound, including the cases where a salt isemployed. For example, a dosage can provide the amount of compoundneeded to provide a serum concentration of the active compound of up tobetween about 1 and 5, 10, 20, 30 or 40 μM. In some embodiments, adosage from about 10 mg/kg to about 50 mg/kg can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg canbe employed for intramuscular injection. In some embodiments, dosagescan be from about 1 umol/kg to about 50 umol/kg, or, optionally, betweenabout 22 umol/kg and about 33 umol/kg of the compound for intravenous ororal administration. An oral dosage form can include any appropriateamount of active material, including for example from 5 mg to, 50, 100,200 or 500 mg per tablet or other solid dosage form.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously, or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having anydesired particle size, and for example, from about 0.01, 0.1 or 0.5 toabout 5, 10, 20 or more microns, and optionally from about 1 to about 2microns. Compounds as disclosed in the present invention havedemonstrated good pharmacokinetic and pharmacodynamics properties, forinstance when administered by the oral or intravenous routes.

The pharmaceutical formulations can comprise an active compounddescribed herein or a pharmaceutically acceptable salt thereof, in anypharmaceutically acceptable carrier. If a solution is desired, water isa carrier of choice for water-soluble compounds or salts. With respectto the water-soluble compounds or salts, an organic vehicle, such asglycerol, propylene glycol, polyethylene glycol, or mixtures thereof,can be suitable. In the latter instance, the organic vehicle can containa substantial amount of water. The solution in either instance can thenbe sterilized in a suitable manner known to those in the art, and forillustration by filtration through a 0.22-micron filter. Subsequent tosterilization, the solution can be dispensed into appropriatereceptacles, such as depyrogenated glass vials. The dispensing isoptionally done by an aseptic method. Sterilized closures can then beplaced on the vials and, if desired, the vial contents can belyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulation is placed in a vial designed formulti-dose use. The pharmaceutical formulations described herein can belyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate may be employed along withvarious disintegrants such as starch (e.g., potato or tapioca starch)and certain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may be also employed as fillers in soft andhard-filled gelatin capsules. Materials in this connection also includelactose or milk sugar as well as high molecular weight polyethyleneglycols. When aqueous suspensions and/or elixirs are desired for oraladministration, the compounds of the presently disclosed subject mattercan be combined with various sweetening agents, flavoring agents,coloring agents, emulsifying agents and/or suspending agents, as well assuch diluents as water, ethanol, propylene glycol, glycerin and variouslike combinations thereof.

In yet another embodiment of the subject matter described herein, thereis provided an injectable, stable, sterile formulation comprising anactive compound as described herein, or a salt thereof, in a unit dosageform in a sealed container. The compound or salt is provided in the formof a lyophilizate, which is capable of being reconstituted with asuitable pharmaceutically acceptable carrier to form a liquidformulation suitable for injection thereof into a subject. When thecompound or salt is substantially water-insoluble, a sufficient amountof emulsifying agent, which is physiologically acceptable, can beemployed in sufficient quantity to emulsify the compound or salt in anaqueous carrier. Particularly useful emulsifying agents includephosphatidyl cholines and lecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired formulation can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles may for example have a particle size in the range of about 0.5to about 10 microns, and optionally from about 0.5 to about 5 microns.The solid particles can be obtained by processing the solid compound ora salt thereof, in any appropriate manner known in the art, such as bymicronization. Optionally, the size of the solid particles or dropletscan be from about 1 to about 2 microns. In this respect, commercialnebulizers are available to achieve this purpose. The compounds can beadministered via an aerosol suspension of respirable particles in amanner set forth in U.S. Pat. No. 5,628,984, the disclosure of which isincorporated herein by reference in its entirety.

When the pharmaceutical formulation suitable for administration as anaerosol is in the form of a liquid, the formulation can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulation sufficiently to result in the formation of droplets withinthe desired size range when subjected to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with subjects (e.g., human subjects) withoutundue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the presently disclosed subject matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the presently disclosedsubject matter. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. In so far as thecompounds of the presently disclosed subject matter are basic compounds,they are all capable of forming a wide variety of different salts withvarious inorganic and organic acids. Acid addition salts of the basiccompounds are prepared by contacting the free base form with asufficient amount of the desired acid to produce the salt in theconventional manner. The free base form can be regenerated by contactingthe salt form with a base and isolating the free base in theconventional manner. The free base forms may differ from theirrespective salt forms in certain physical properties such as solubilityin polar solvents.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metal hydroxides, orof organic amines. Examples of metals used as cations, include, but arenot limited to, sodium, potassium, magnesium, calcium, and the like.Examples of suitable amines include, but are not limited to,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, and procaine.

The base addition salts of acidic compounds are prepared by contactingthe free acid form with a sufficient amount of the desired base toproduce the salt in the conventional manner. The free acid form can beregenerated by contacting the salt form with an acid and isolating thefree acid in a conventional manner. The free acid forms may differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

Synthesis of Tricyclic Lactams

Tricyclic lactams of the present invention can be synthesized by anymeans known to those of ordinary skill in the art, including forexample, according to the generalized Schemes of 1 through 11 below.

A method for the preparation of substituted tricyclic lactams isprovided that includes efficient methods for the preparation of atricyclic lactam ring system and subsequent displacement of an arylsulfone with an amine.

In Scheme 1, diethyl succinate is employed to prepare the pyrimidineester, 2, according to the method of A. Haidle, See, WO 2009/152027entitled 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives forMARK inhibition. The ester intermediate 2 can be reduced by directlyreacting the ester with a reducing agent such as lithium borohydride ina protic organic solvent such as ethanol to produce the correspondingprimary alcohol. The primary alcohol can be reacted with a reagent suchas phosphorus tribromide in an organic solvent such as dimethylforamideto produce the primary bromide 3. The primary bromide 3 can be condensedwith the lactam 4 optionally at low temperature using a base such aslithium diisopropylamide in an organic solvent such as tetrahydrofuranto produce the lactam 5. Lactam 5 can be deprotected by directlyreacting Compound 5 with an aqueous acid such as HCl=pH 1 solution.Lactam 6 can be directly reacted with an organic base such as1,8-diazabicyclo[5.4.0]undec-7-ene in a protic solvent such as ethanoloptionally at high temperature to cyclize Compound 5 to form thetricyclic lactam 7. The thiol moiety can be subsequently oxidized to thesulfone 8 by directly reacting Compound 7 with an oxidizing reagent suchas meta-chloroperoxybenzoic acid. The sulfone, 8, can be directlyreacted with an amine, 9, in the presence of a strong base such aslithium hexamethyldisilazane to form the tricyclic lactam 10.

In Scheme 2, the tricyclic lactam 7 is directly reacted with anoxidizing reagent such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ) to form the alkene 11. Alkene 11 can be directly reacted with anoxidizing reagent such as meta-chloroperoxybenzoic acid to form thesulfone intermediate 12. The sulfone, 12, can be condensed with anamine, 13, in the presence of a strong base such as lithiumhexamethyldisilazane to form the tricyclic lactam 14.

Scheme 3 illustrates the synthesis of a di-protected lactam useful inthe preparation of tricyclic lactams. Compound 15 is prepared accordingto the method of Arigon, J., See, US 2013/0289031 entitled Pyrimidinonederivatives, preparation thereof and pharmaceutical use thereof.Compound 15 is protected with a suitable protecting group by directlyreacting Compound 15 with di-tert-butyl carbonate (Boc anhydride) in thepresence of an organic base such as triethylamine ordiisopropylethylamine in an organic solvent such as dichloromethane toform the protected amine 16. The protected amine 16 can be directlyreacted with methyl chloroacetate in the presence of a base such aspotassium carbonate in an organic solvent such as acetonitrile to formthe ester 17. The ester 17 can be cyclized by directly reacting theester with an acid such as hydrochloric acid in a protic solvent such asmethanol optionally at a high temperature to form the spirolactam 18.The lactam 18 can be directly reacted with a protecting reagent such aschloromethyl methyl ether (MOM-Cl) in the presence of an organic basesuch as diisopropylethylamine in an organic solvent such asdichloromethane optionally at a low or at ambient temperature to formthe MOM-protected amine 19. The lactam 19 can be protected by directlyreacting the lactam with a suitable protecting reagent such aschloromethyl methyl ether (MOM-Cl) in the presence of a base such assodium bis(trimethylsilyl)amide in an organic solvent such astetrahydrofuran optionally at a low temperature. Additional lactamintermediates such as Compounds 25 and 31 can be synthesized usinganalogous chemistry as described for the synthesis of Compound 4. Thechemistry for the production of Compounds 25 and 31 is illustrated inSchemes 5 and 6.

Scheme 4 illustrates the coupling of a tricyclic lactam sulfone with anamine to generate compounds of Formula I, II, III, and IV.

Scheme 7 illustrates the preparation of the tricyclic lactam compound33. Compound 32 is prepared according to the method of Tavares, See,U.S. Pat. No. 8,598,186. Compound 32 is directly reacted with sulfone 8optionally in the presence of an organic base such as lithiumhexamethyldisilazane and the amine 32 to form the amine 33. The samechemistry can be employed to produce the alkene compound 34.

In one embodiment a lactam intermediate is treated with BOC-anhydride inthe presence of an organic base such as triethylamine in an organicsolvent such as dichloromethane. The Boc protected lactam is treatedwith carbon dioxide in the presence of a nickel catalyst to generate acarboxylic acid. The carboxylic acid is reacted with thionyl chloride inthe presence of an organic solvent such as toluene. The resulting acidchloride is treated with an amine to generate an amide that can bedeprotected with a strong acid such as trifluoroacetic acid to generatethe final target compound.

Alternatively, the lactam can be generated by reacting the carboxylicacid with a protected amine in the presence of a strong acid and adehydrating agent, which can be together in one moiety as a strong acidanhydride. Examples of strong acid anhydrides include, but are notlimited to, trifluoroacetic acid anhydride, tribromoacetic acidanhydride, trichloroacetic acid anhydride, or mixed anhydrides. Thedehydrating agent can be a carbodiimide based compound such as but notlimited to DCC (N,N-dicyclohexylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC(N,N-diisopropylcarbodiimide). An additional step may be necessary totake off the N-protecting group and the methodologies are known to thoseskilled in the art

Alternatively, the SMe moiety bonded to the pyrimidine ring can besubstituted with any leaving group that can be displaced by a primaryamine, for example to create an intermediate for a final product such asBr, I, F, SO₂Me, SOalkyl, SO₂alkyl. See, for Example, PCT /US2013/037878to Tavares.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol

4-Chloro-2-methylsulfanyl-5-pyrimidinecarboxylate ethyl ester (62 g, 260mmol) was dissolved in anhydrous tetrahydrofuran (500 mL) in a 3-necked5 L round bottomed flask fitted with a mechanical stirrer, additionfunnel, temperature probe and nitrogen inlet. The solution was cooled to0° C. Diisobutylaluminum hydride in tetrahydrofuran (1M solution, 800mL) was added dropwise over a period of 2 hours. After the addition wascomplete, the reaction mixture was kept at 0° C. for 0.5 hours. Thereaction was quenched at 0° C. by the slow addition of saturated aqueoussodium sulfate (265.3 mL, 530.7 mmol) keeping the internal reactiontemperature below 10° C. Ethyl acetate (900 mL) was added and thereaction slowly warmed to room temperature overnight. 6M HCl was addedtill the reaction mixture was slightly acidic (pH 6). The reactionmixture was filtered thru a pad of Celite® and the aluminum salts werewashed with ethyl acetate (1 L). The filtrate was poured into aseparatory funnel and washed twice with water (600 mL) and finally withbrine (600 mL). The organic layer was dried over sodium sulfate,filtered thru Celite® and the solvent concentrated in vacuo to afford39.2 g (77% crude yield) of a dark yellow oil. The material was used asis for the next step. NMR (CDCl₃) δ 8.56 (s, 1H), 4.76 (s, 2H), 2.59 (s,3H); MS (ESI+) for C₆H₇ClN₂OS m/z 191.0 (M+H)⁺.

4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde

[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol (39.2 g, 206 mmol) wastaken up in methylene chloride (520 mL) at room temperature.Manganese(IV) oxide (140 g, 1.60 mol) was added in one portion and thereaction mixture stirred at room temperature overnight. The reactionmixture was filtered through a pad of Celite® and washed with methylenechloride. The filtrate was concentrated under reduced pressure to afforda dark yellow semisolid. The crude product was purified by reverse phasechromatography running a gradient of 1:9 acetonitrile:water (0.1% TFA)to 100% acetonitrile (0.1% TFA). The desired fractions were combined andthe acetonitrile was removed under reduced pressure causingprecipitation of the desired product. The solids were removed byfiltration and the solids washed with water and dried under vacuum at50° C. Affords 16.6 g (43% yield) of the desired product as a whitesolid. NMR (CDCl₃) δ 10.32 (s, 1H), 8.88 (s, 1H), 2.65 (s, 3H); MS(ESI+) for C₆H₅ClN₂OS m/z 189.0 (M+H)⁺.

General Procedure A.

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Isopropylmagnesium chloride:lithium chloride complex (4.43 g, 30.5 mmol,25.4 mL of a 1.2M solution) was added to tetrahydrofuran (104 mL) in a500 mL round bottomed flask which had been flame dried and cooled underArgon. The solution was cooled to −15° C. Ethyl propiolate (3.26 mL,32.1 mmol) was added dropwise affording a yellow solution. Stirring wascontinued at −15° C. for 30 minutes and then4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde (6.06 g, 32.1 mmol) intetrahydrofuran (52 mL) was added rapidly. After 10 minutes, thereaction was quenched by the addition of saturated aqueous ammoniumchloride (40 mL). The reaction mixture was warmed to room temperatureand poured into a separatory funnel partitioning between ethyl acetate(200 mL) and water (100 mL). The organic layer removed and the aqueouslayer extracted with ethyl acetate (100 mL). The combined organic layerswere washed with brine (100 mL), dried over sodium sulfate, filtered andthe solvent removed in vacuo to afford a dark red oil. The product waspurified by silica gel chromatography using a gradient of 1:4 to 2:3ethyl acetate:hexanes which afforded 3.76 g (37% yield) of the desiredproduct as a light red oil. NMR (CDCl₃) δ 8.75 (s, 1H), 5.81 (d, 1H,J=6.0 Hz), 2.72 (bs, 1H), 2.60 (s, 3H), 1.33 (t, 3H, J=7.2 Hz); MS(ESI+) for C₁₁H₁₁ClN₂O₃S m/z 287.9 (M+H)⁺.

tert-Butyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Following General Procedure A and using tert-butyl propiolate affordsthe desired product in 74% yield as a viscous yellow oil. NMR (CDCl₃) δ8.75 (s, 1H), 5.79 (d, 1H, J=5.4 Hz), 4.27 (q, 2H, J=7.2 Hz), 2.97 (d,1H, J=5.4 Hz), 2.60 (s, 3H), 1.52 (s, 9H); MS (ESI+) for C₁₃H₁₅ClN₂O₃Sm/z 314.9 (M+H)⁺.

General Procedure B.

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate(3.40 g, 11.8 mmol) was taken up in 1,4-Dioxane (100 mL) at roomtemperature under argon. Triethylamine (3.3 mL, 24 mmol) was added andthe mixture heated to 60° C. for 1 h. The reaction mixture was cooled toroom temperature and the solvent removed in vacuo. The resultant darkorange oil was re-evaporated twice with toluene. Affords the desiredproduct (3:1 E:Z double bond isomers) in 99% yield as a dark orange oil.NMR (CDCl₃) (major E isomer) δ 8.69 (s, 1H), 7.65 (d, 1H, J=18.0 Hz),6.81 (d, 1H, J=18.0 Hz), 4.32 (q, 2H, J=6.0 Hz), 2.64 (s, 3H), 1.36 (t,3H, J=6.0 Hz); NMR (CDCl₃) (minor Z isomer) δ 8.87 (s, 1H), 6.89 (d, 1H,J=12.0 Hz), 6.23 (d, 1H, J=12.0 Hz), 4.14 (q, 2H, J=6.0 Hz), 2.63 (s,3H), 1.23 (t, 3H, J=6.0 Hz); MS (ESI+) for C₁₁H₁₁ClN₂O₃S m/z 287.0(M+H)⁺.

tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate

Isomerization of tert-butyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate usingGeneral Procedure B affords the desired product (5:1 E:Z double bondisomers) in a 99% yield as a viscous dark yellow oil. NMR (CDCl₃) (majorE isomer) δ 8.67 (s, 1H), 7.54 (d, 1H, J=15.6 Hz), 6.72 (d, 1H, J=15.6Hz), 2.64 (s, 3H), 1.54 (s, 9H); NMR (CDCl₃) (minor Z isomer) δ 8.86 (s,1H), 6.76 (d, 1H, J=12.0 Hz), 6.18 (d, 1H, J=12.0 Hz), 2.63 (s, 3H),1.40 (s, 9H); MS (ESI+) for C₁₃H₁₅ClN₂O₃S m/z 314.9 (M+H)⁺.

General Procedure C.

Ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate (3.40g, 11.8 mmol) was taken up in acetonitrile (20 mL) at room temperature.1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine (3.86 g, 14.2 mmol)was added followed by triethylamine (3.30 mL, 23.7 mmol). The mixturewas stirred at room temperature overnight. The reaction mixture wastransferred to a separatory funnel transferring with ethyl acetate (250mL). The organic layer was washed twice with a 10% citric acid (aq) (20mL)/brine (60 mL) mixture. The organic layer was dried over sodiumsulfate, filtered and the solvent removed in vacuo to afford a yellowoil. The product was purified by silica gel chromatography using agradient from 1:9 to 2:3 ethyl acetate:hexanes which afforded 2.48 g(41% yield) of the desired product as a pale yellow oil. NMR (CDCl₃) δ8.58 (s, 1H), 4.72 (m, 1H), 4.46 (m, 1H), 4.16 (q, 2H, J=6.9 Hz), 3.52(m, 1H), 2.96 (m, 2H), 2.54 (s, 3H), 2.31 (m, 3H), 1.81-1.52 (m, 5H),1.24 (t, 3H, J=6.9 Hz) 1.08-0.96 (m, 21H); MS (ESI+) for C₂₆H₄₃N₃O₄SSim/z 522.2 (M+H)⁺.

Ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and1-{[(triisopropylsilyl)oxy]methyl}cyclohexanamine using GeneralProcedure C afforded the desired product in 17% yield as a pale yellowoil. NMR (CDCl₃) δ 8.63 (s, 1H), 4.83 (m, 1H), 4.73 (m, 1H), 4.14 (q,2H, J=6.0 Hz), 3.86 (m, 1H), 2.97 (m, 2H), 2.55 (s, 3H), 1.97 (m, 1H),1.72-1.48 (m, 9H), 1.23 (t, 3H, J=6.0 Hz), 1.13-0.95 (m, 21H); MS (ESI+)for C₂₇H₄₅N₃O₄SSi m/z 536.2 (M+H)⁺.

Ethyl8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and1-{[tert-butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine using GeneralProcedure C afforded the desired product in 52% yield as a pale yellowoil. NMR (CDCl₃) δ 8.62 (s, 1H), 4.94 (m, 1H), 4.18 (m, 3H), 3.63 (m,1H), 2.93 (m, 2H), 2.57 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H), 1.23 (t,3H, J=7.2 Hz), 0.89 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H); MS (ESI+) forC₂₁H₃₅N₃O₄SSi m/z 454.3 (M+H)⁺.

Ethyl2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and2-[(triisopropylsilyl)oxy]ethanamine using General Procedure C affordedthe desired product in 45% yield as a pale yellow oil. NMR (CDCl₃) δ8.60 (s, 1H), 4.72 (m, 1H), 4.63 (m, 1H), 4.19 (q, 2H, J=6.0 Hz), 3.97(m, 2H), 3.22 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.28 (t, 3H, J=6.0Hz), 1.17-1.02 (m, 21H); MS (ESI+) for C₂₂H₃₇N₃O₄SSi m/z 468.1 (M+H)⁺.

tert-Butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of tert-Butyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 0.5 Mammonia/dioxane using General Procedure C afforded the desired productin 60% yield as an off-white solid. NMR (CDCl₃) δ 8.66 (s, 1H), 6.18(bs, 1H), 4.34 (m, 1H), 3.05-2.80 (m, 2H), 2.56 (s, 3H), 1.51 (s, 9H);MS (ESI+) for C₁₃H₁₇N₃O₃S m/z 296.0 (M+H)⁺.

General Procedure D.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate(2.48 g, 4.75 mmol) was taken up in tetrahydrofuran (10 mL) andacetonitrile (10 mL) at room temperature. 1M Sodium hydroxide (10 mL, 10mmol) was added at room temperature for 1 hour. The reaction wasquenched by the addition of 10% citric acid till pH ca 6-7. The reactionmixture was transferred to a separatory funnel with water (30 mL) andethyl acetate (150 mL). The aqueous layer was removed and the organiclayer washed with brine (50 mL). The organic layer was dried over sodiumsulfate, filtered and the solvent concentrated in vacuo to afford the2.08 g (89% yield) of the desired product as a dark yellow oil. NMR(CDCl₃) δ 8.46 (s, 1H), 4.58 (m, 1H), 4.39 (m, 1H), 3.71 (m, 1H), 2.88(m, 2H), 2.51 (s, 3H), 2.26 (m, 3H), 1.97-1.45 (m, 6H) 1.12-0.92 (m,21H); MS (ESI+) for C₂₄H₃₉N₃O₄SSi m/z 494.2 (M+H)⁺.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Saponification of ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateusing General Procedure D affords the desired product in 95% yield as adark yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 4.75 (m, 1H), 4.53 (m, 1H),3.96 (m, 1H), 2.99 (m, 2H), 2.54 (s, 3H), 1.93-1.48 (m, 10H), 1.13-0.95(m, 21H); MS (ESI+) for C₂₅H₄₁N₃O₄SSi m/z 508.1 (M+H)⁺.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Saponification of ethyl8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateusing General Procedure D affords the desired product in 99% yield as apale yellow foam. NMR (CDCl₃) δ 8.69 (s, 1H), 4.88 (m, 1H), 4.51 (m,1H), 3.82 (m, 1H), 3.17 (m, 1H), 2.79 (m, 1H), 2.57 (s, 3H), 1.65 (s,3H), 1.60 (s, 3H), 0.92 (s, 9H), 0.11 (s, 6H); MS (ESI+) forC₁₉H₃₁N₃O₄SSi m/z 426.3 (M+H)⁺.

2-(Methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Saponification of ethyl2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateusing General Procedure D affords the desired product in 98% yield as anorange foam. NMR (CDCl₃) δ 8.56 (s, 1H), 4.71 (m, 1H), 4.54 (m, 1H),3.99 (m, 2H), 3.32 (m, 1H), 3.01 (m, 2H), 2.53 (s, 3H), 1.16-0.98 (m,21H); MS (ESI+) for C₂₀H₃₃N₃O₄SSi m/z 440.2 (M+H)⁺.

2-(Methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

tert-Butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate(220 mg, 0.74 mmol) was taken up in trifluoroacetic acid (5 mL) at roomtemperature under argon. The mixture was stirred at room temperature for45 minutes. The solvent was removed in vacuo to a pink oil which wasre-evaporated first from toluene and finally methanol affording 180 mg(99% yield) of the desired product as an off-white solid. NMR (MeOH-d₄)δ 8.49 (s, 1H), 4.59 (m, 1H), 3.16-2.91 (m, 2H), 2.63 (s, 3H); MS (ESI+)for C₉H₉N₃O₃S m/z 240.0 (M+H)⁺.

General Procedure E.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid (2.08 g, 4.21 mmol) was taken up in N,N-dimethylformamide (30 mL)at room temperature. 1-(2,4-dimethoxyphenyl)methanamine (1.26 mL, 8.42mmol) was added followed by the addition ofN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (4.80 g, 12.6 mmol) and N,N-diisopropylethylamine(4.40 mL, 25.3 mmol). The reaction mixture was stirred overnight at roomtemperature. The product was diluted with water (50 mL) and poured intoa separatory funnel. The mixture was extracted with twice with ethylacetate (150 mL) and the combined organic layers were thrice washed withhalf-saturated aqueous LiCl (20 mL). The combined organic layers weredried over sodium sulfate, filtered and the solvent removed in vacuo toafford a dark yellow oil. The product was purified by silica gelchromatography using a gradient from 1:4 to 2:3 ethyl acetate:hexaneswhich afforded 2.39 g (88% yield) of the desired product as a brownsticky solid. NMR (CDCl₃) δ 8.58 (s, 1H), 7.07 (m, 1H), 6.62 (m, 1H),6.39 (m, 2H), 4.56 (m, 1H), 4.38 (m, 2H), 4.20 (m, 1H), 3.80 (s, 3H),3.66 (s, 3H), 3.48 (m, 1H), 3.02 (m, 2H), 2.55 (s, 3H), 2.35 (m, 2H),2.06 (m, 1H), 1.70-1.31 (m, 5H), 1.09-0.90 (m, 21H); MS (ESI+) forC₃₃H₅₀N₄O₅SSi m/z 643.2 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 52% yieldas a yellow solid. NMR (CDCl₃) δ 8.60 (s, 1H), 6.87 (m, 2H), 6.37 (m,2H), 4.71 (m, 1H), 4.49-4.14 (m, 4H), 3.79 (s, 3H), 3.70 (s, 3H), 3.18(m, 1H), 2.86 (m, 1H), 2.65 (m, 1H), 2.54 (s, 3H), 2.20 (m, 1H), 1.98(m, 2H), 1.61-1.48 (m, 6H), 1.08-0.92 (bs, 21H); MS (ESI+) forC₃₄H₅₂N₄O₅SSi m/z 657.2 (M+H)⁺.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 86% yieldas a viscous yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 6.95 (m, 1H), 6.80(m, 1H), 6.37 (m, 2H), 4.71 (m, 1H), 4.27 (m, 2H), 4.13 (m, 1H), 3.84(m, 1H), 3.79 (s, 3H), 3.70 (s, 3H), 3.15 (m, 1H), 2.77 (m, 1H), 2.56(s, 3H), 1.64 (s, 3H), 1.58 (s, 3H), 0.85 (s, 9H), 0.02 (s, 3H), −0.03(s, 3H); MS (ESI+) for C₂₈H₄₂N₄O₅SSi m/z 575.4 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 57% yieldas a dark yellow solid. NMR (CDCl₃) δ 8.59 (s, 1H), 7.06 (m, 1H), 6.39(m, 3H), 4.51 (m, 2H), 4.32 (m, 2H), 3.93 (m, 2H), 3.81 (s, 3H), 3.73(s, 3H), 3.18 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.11-0.95 (m, 21H);MS (ESI+) for C₂₉H₄₄N₄O₅SSi m/z 589.4 (M+H)⁺.

2-(Methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 94% yieldas a dark yellow oil. NMR (CDCl3) δ 8.64 (s, 1H), 6.09 (bs, 1H), 6.04(bs, 1H), 4.25 (m, 1H), 3.68 (m, 2H), 2.86 (m, 2H), 2.54 (s, 3H),2.07-1.54 (m, 8H), 1.33-0.96 (m, 21H); MS (ESI+) for C₂₄H₄₀N₄O₃SSi m/z493.1 (M+H)⁺.

General Procedure F.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide(2.39 g, 3.72 mmol) was taken up in tetrahydrofuran (42 mL) at roomtemperature. Tetra-n-butylammonium fluoride (5.6 mL, 5.6 mmol, 1Msolution in THF) was added and the reaction stirred for 10 minutes atroom temperature. The reaction mixture was concentrated in vacuo to anorange oil and was transferred to a separatory funnel and partitionedbetween ethyl acetate (200 mL) and water (50 mL). The aqueous layer wasremoved and the organic layer washed with water (50 mL) and brine (50mL). The organic layer was dried over sodium sulfate, filtered and thesolvent removed in vacuo to afford a dark yellow semi-solid. The productwas purified by reverse phase chromatography using a gradient from 1:9to 3:2 acetonitrile:water (0.1% TFA). Lyophilization of the desiredfractions afforded 1.81 g (99% yield) of the desired product as a darkyellow powder. NMR (CDCl₃) δ 8.59 (s, 1H), 7.21 (m, 1H), 7.02 (m, 1H),6.39 (m 2H), 4.56 (m, 1H), 4.29 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H),3.70 (m, 2H), 3.41 (m, 1H), 3.20 (m, 1H), 2.87 (m, 1H), 2.55 (s, 3H),2.18 (m, 1H), 1.97-1.59 (m, 7H); MS (ESI+) for C₂₄H₃₀N₄O₅S m/z 487.1(M+H)⁺.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation ofN-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 97% yield as ayellow powder. NMR (CDCl₃) δ 8.69 (s, 1H), 7.58 (m, 1H), 7.07 (m, 1H),6.48 (m, 2H), 4.72 (m, 2H), 4.38 (m, 2H), 3.89 (s, 3H), 3.87 (s, 3H),3.31 (m, 1H), 2.99 (m, 1H), 2.81 (m, 1H), 2.64 (s, 3H), 2.11 (m, 3H),1.94-1.58 (m, 7H); MS (ESI+) for C₂₅H₃₂N₄O₅S m/z 501.1 (M+H)⁺.

8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 98% yield as ayellow powder. NMR (CDCl₃) δ 8.61 (s, 1H), 7.51 (m, 1H), 7.01 (m, 1H),6.37 (m, 2H), 4.72 (m, 1H), 4.65 (m, 1H), 4.26 (m, 2H), 3.80 (s, 3H),3.76 (s, 3H), 3.63 (m, 1H), 3.18 (m, 1H), 2.80 (m, 1H), 2.57 (s, 3H),1.58 (s, 3H), 1.56 (s, 3H); MS (ESI+) for C₂₂H₂₈N₄O₅S m/z 461.4 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 97% yield as apale yellow powder. NMR (CDCl₃) δ 8.59 (s, 1H), 7.02 (m, 1H), 6.69 (m,1H), 6.43 (m, 2H), 4.34 (m, 3H), 3.88 (m, 4H), 3.81 (s, 3H), 3.78 (s,3H), 3.02 (m, 2H), 2.55 (s, 3H); MS (ESI+) for C₂₀H₂₄N₄O₅S m/z 432.9(M+H)⁺.

N-[1-(Hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of2-(methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 31% yield asan off white powder. NMR (CDCl₃) δ 8.66 (s, 1H), 6.19 (bs, 1H), 5.97(bs, 1H), 4.31 (m, 1H), 3.69 (s, 2H), 2.92 (m, 2H), 2.56 (s, 3H),1.95-1.65 (m, 9H); MS (ESI+) for C₁₅H₂₀N₄O₃S m/z 337.0 (M+H)⁺.

General Procedure G.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide(1.81 g, 3.72 mmol) was taken up in methylene chloride (30 mL) at roomtemperature. Triethylamine (1.3 mL, 9.3 mmol) was added followed by therapid addition of methanesulfonyl chloride (0.43 mL, 5.6 mmol). Thereaction mixture was stirred at room temperature for 30 minutes beforebeing heated at reflux overnight. The solvent was removed in vacuoaffording a brown semi-solid. The product was purified by reverse phasechromatography using a gradient from 1:9 to 3:2 acetonitrile:water (0.1%TFA). Lyophilization of the desired fractions gave 764 mg (44% yield) ofthe desired product as a light brown powder. NMR (CDCl₃) δ 8.49 (s, 1H),7.13 (m, 1H), 6.43 (m, 2H), 5.49 (m, 1H), 4.37-4.16 (m, 4H), 3.81 (s,3H), 3.80 (s, 3H), 3.29 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H), 2.41 (m,1H), 1.99-1.52 (m, 7H); MS (ESI+) for C₂₄H₂₈N₄O₄S m/z 469.1 (M+H)⁺.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Mesylation and cyclization ofN-(2,4-dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure G afforded the desired product in 73% yield as awhite powder. NMR (MeOH-d₄) δ 8.48 (s, 1H), 7.14 (m, 1H), 6.43 (m, 1H),6.39 (m, 1H), 5.61 (m, 1H), 4.31 (m, 1H), 4.21 (m, 2H), 3.83 (s, 3H),3.80 (s, 3H), 3.38 (m, 1H), 3.22 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H),2.22 (m, 1H), 1.99-1.09 (m, 9H); MS (ESI+) for C₂₅H₃₀N₄O₄S m/z 483.1(M+H)⁺.

8-(2,4-Dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Mesylation and cyclization of8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure G afforded the desired product in 82% yield as ayellow powder. NMR (CDCl₃) δ 8.45 (s, 1H), 7.14 (m, 1H), 6.40 (m, 2H),5.56 (m, 1H), 4.40 (m, 1H), 4.31 (m, 2H), 4.13 (m, 1H), 3.81 (s, 3H),3.79 (s, 3H), 3.28 (m, 1H), 2.88 (m, 1H), 2.79 (s, 3H), 1.80 (s, 3H),1.45 (s, 3H); MS (ESI+) for C₂₂H₂₆N₄O₄S m/z 443.5 (M+H)⁺.

8-(2,4-Dimethoxybenzyl)-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Mesylation and cyclization ofN-(2,4-dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure G afforded the desired product in 81% yield as alight brown powder. NMR (CDCl₃) δ 8.52 (s, 1H), 7.11 (m, 1H), 6.44 (m,2H), 5.31 (m, 1H), 4.68-4.29 (m, 5H), 4.04 (m, 1H), 3.81 (s, 3H), 3.80(s, 3H), 3.20 (m, 1H), 3.01 (m, 1H), 2.82 (s, 3H); MS (ESI+) forC₂₀H₂₂N₄O₄S m/z 415.0 (M+H)⁺.

General Procedure H.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione(764 mg, 1.63 mmol) was taken up in trifluoroacetic acid (10 mL) at roomtemperature under argon. The mixture was heated to 75° C. for 6 hours,cooled to room temperature and left to stir overnight. The solvent wasremoved in vacuo to afford a purple oil. The product was purified byreverse phase chromatography using a gradient from 100% water (0.1% TFA)to 1:1 acetonitrile:water (0.1% TFA). Lyophilization of the desiredfractions afforded 117 mg (23% yield) of the desired product as a paleyellow powder. NMR (CDCl₃) δ 8.52 (s, 1H), 5.58 (m, 2H), 4.34 (bs 2H),3.29 (m, 1H), 3.06 (m, 1H), 2.84 (s, 3H), 2.48 (m, 1H), 2.31 (m, 1H),2.11-1.65 (m, 6H); MS (ESI+) for C₁₅H₁₈N₄O₂S m/z 319.0 (M+H)⁺.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Removal of the dimethoxybenzyl group of8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dioneusing General Procedure H afforded the desired product in 23% yield as awhite powder. NMR (MeOH-d₄) δ 8.60 (s, 1H), 4.78 (m, 1H), 4.54 (m, 2H),3.38 (m, 1H), 2.86 (s, 3H), 2.84 (m, 1H), 2.12-1.30 (m, 10H); MS (ESI+)for C₁₆H₂₀N₄O₂S m/z 333.1 (M+H)⁺.

10,10-Dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of8-(2,4-dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneusing General Procedure H afforded the desired product in 56% yield as awhite powder. NMR (CDCl₃) δ 8.54 (s, 1H), 5.65 (m, 1H), 5.51 (bs, 1H),4.31 (s, 2H), 3.23 (m, 1H), 3.04 (m, 1H), 2.85 (s, 3H), 1.78 (s, 3H),1.68 (s, 3H); MS (ESI+) for C₁₃H₁₆N₄O₂S m/z 293.2 (M+H)⁺.

2-(Methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of8-(2,4-dimethoxybenzyl)-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneusing General Procedure H afforded the desired product in 21% yield as abrown powder. NMR (MeOH-d₄) δ 8.65 (s, 1H), 4.78-4.05 (m, 5H), 3.32 (m,2H), 3.01 (m, 1H), 2.87 (s, 3H); MS (ESI+) for C₁₁H₁₂N₄O₂ m/z 265.0(M+H)⁺.

General Procedure I.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione(117 mg, 0.367 mmol) was taken up in N,N-dimethylacetamide (4.0 mL, 43mmol) at room temperature under argon.5-(4-methylpiperazin-1-yl)pyridin-2-amine (100 mg, 0.55 mmol) was addedand the reaction mixture was heated just to 150° C. and then immediatelyremoved from the heat and cooled to room temperature. The product waspurified by reverse phase chromatography using a gradient from 100%Water (0.1% TFA) to 1:1 acetonitrile:water (0.1% TFA). Lyophilization ofthe desired fractions afforded 11 mg (7% yield) of the desired productas an orange powder. NMR (MeOH-d₄) δ 8.54 (s, 1H); 8.06 (m, 1H), 7.85(m, 1H), 7.69 (m, 1H), 4.69 (m, 1H), 4.39 (m, 2H), 3.95 (m, 2H), 3.69(m, 2H), 3.39-3.15 (m, 5H), 3.02 (s, 3H), 2.77 (m, 1H), 2.21 (m, 1H),2.09-1.67 (m, 7H); MS (ESI+) for C₂₄H₃₀N₈O₂ m/z 463.1 (M+H)⁺.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

S_(N)Ar reaction using General Procedure I and2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dioneafforded the desired product in 38% yield as a yellow powder. NMR(MeOH-d₄) δ 8.52 (s, 1H), 8.07 (m, 1H), 7.89 (bs, 1H), 7.71 (m, 1H),4.71 (m, 1H), 4.40 (m, 2H), 3.94 (m, 2H), 3.68 (m, 2H), 3.35-3.22 (m,5H), 3.02 (s, 3H), 2.73 (m, 1H), 2.02-1.25 (m, 10H); MS (ESI+) forC₂₅H₃₂N₈O₂ m/z 477.2 (M+H)⁺.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

S_(N)Ar reaction using General Procedure I and10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneafforded the desired product in 10% yield as a yellow powder. NMR(MeOH-d₄) δ 8.52 (s, 1H), 8.05 (m, 2H), 7.86 (m, 1H), 7.67 (m, 1H), 4.66(m, 1H), 4.33 (m, 2H), 3.93 (m, 2H), 3.68 (m, 2H), 3.38-3.21 (m, 5H),3.01 (s, 3H), 2.72 (m, 1H), 1.65 (s, 3H), 1.54 (s, 3H); MS (ESI+) forC₂₂H₂₈N₈O₂ m/z 437.4 (M+H)⁺.

2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

S_(N)Ar reaction using General Procedure I and2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneafforded the desired product in 15% yield as an orange powder. NMR(DMSO-d₆) δ 8.41 (s, 1H), 8.05 (m, 1H), 7.93 (m, 1H), 7.82 (m, 1H),4.59-4.34 (m, 3H), 4.03-3.84 (m, 4H), 3.49 (m, 2H), 3.30-3.09 (m, 6H),2.80 (s, 3H), 2.80-2.67 (m, 2H); MS (ESI+) for C₂₀H₂₄N₈O₂ m/z 409.1(M+H)⁺.

General Procedure J.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydro-7′H-dispiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5′,2″-[1,3]dithian]-7′-one

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione(90.0 mg, 0.189 mmol) and 1,3-propanedithiol (0.0379 mL, 0.378 mmol)were taken up in toluene (5 mL) at room temperature under argon.p-Toluenesulfonic acid (0.02 g, 0.1 mmol) was then added. The reactionvessel was fitted with a condenser and the reaction mixture heated atreflux overnight. The reaction mixture was cooled to room temperatureand the solvent removed in vacuo affording a thick dark yellow oil. Theproduct was purified by reverse phase chromatography using a gradientfrom 100% water (0.1% TFA) to 3:2 acetonitrile:water (0.1% TFA).Lyophilization of the desired fractions afforded 35 mg (33% yield) ofthe desired product as a pale yellow powder. NMR (MeOH-d₄) δ 8.52 (s,1H), 7.90 (m, 1H), 7.84 (m, 1H), 7.52 (m, 1H), 4.64 (m, 1H), 4.53 (m,1H), 4.16 (m, 1H), 3.60 (m, 2H), 3.41-3.26 (m, 6H), 3.01 (s, 3H), 2.91(m, 1H), 2.75 (m, 1H), 2.61 (m, 1H), 2.21 (m, 1H), 2.11 (m, 1H),1.95-1.72 (m, 10H), 1.61 (m, 1H), 1.33 (m 2H); MS (ESI+) for C₂₈H₃₈N₈OS₂m/z 567.1 (M+H)⁺.

10′,10′-Dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(8′H)-one

Dithiane formation using General Procedure J and10,10-dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneafforded the desired product in 43% yield as an orange powder. NMR(MeOH-d₄) δ 8.53 (s, 1H), 7.88 (m, 1H), 7.82 (m, 1H), 7.47 (m, 1H), 4.47(m, 1H), 4.41 (m, 1H), 4.16 (m, 1H), 3.92-3.15 (m, 11H), 3.00 (s, 3H),2.90-2.81 (m, 3H), 2.21 (m, 1H), 1.87 (m, 1H), 1.60 (s, 3H), 1.48 (s,3H); MS (ESI+) for C₂₅H₃₄N₈OS₂ m/z 527.1 (M+H)⁺.

General Procedure K.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(5′H)-one

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydro-7′H-dispiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5′,2″-[1,3]dithian]-7′-one(35 mg, 0.062 mmol) in ethanol (1 ml) was added to Raney nickel (1 mL ofthe aqueous slurry which was washed thrice with ethanol decanting offthe ethanol after each washing) in ethanol (3 mL) under argon. Thereaction mixture was heated to 45° C. for 30 minutes. After cooling toroom temperature, the reaction mixture was filtered through a pad ofCelite® washing with ethanol. The solvent was removed in vacuo affordinga yellow oil. The product was purified by reverse phase chromatographyusing a gradient from 100% water (0.1% TFA) to 3:2 acetonitrile:water(0.1% TFA). Lyophilization of the desired fractions afforded 4 mg (14%yield) of the desired product as a pale yellow powder. NMR (CDCl₃) δ7.95 (m, 1H), 7.93 (s, 1H), 7.77 (m, 1H), 7.51 (m, 1H), 4.57 (m, 1H),4.51 (m, 1H), 4.35 (m, 1H), 3.89 (m, 2H), 3.69 (m, 2H), 3.37 (m, 2H),3.15 (m, 2H), 3.01 (s, 3H), 2.79 (m, 1H), 2.58 (m, 1H), 2.39 (m, 1H),2.05-1.45 (m, 11H); MS (ESI+) for C₂₅H₃₄N₈O m/z 463.1 (M+H)⁺.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin-7(8H)-one

Desulfurization using General Procedure K and10′,10′-dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(8′H)-oneafforded the desired product in 11% yield as a yellow powder. NMR(MeOH-d₄) δ 7.93 (m, 2H), 7.75 (m, 1H), 7.43 (m, 1H), 4.52 (m, 1H), 4.32(m, 2H), 3.89 (m, 2H), 3.67 (m, 2H), 3.38 (m, 2H), 3.14 (m, 2H), 3.01(s, 3H), 2.79 (m, 1H), 2.60 (m, 1H), 2.39 (m, 1H), 2.00 (m, 1H), 1.55(s, 3H), 1.54 (s, 3H); MS (ESI+) for C₂₂H₃₀N₈O m/z 423.1 (M+H)⁺.

General Procedure L.

(1-Aminocyclohexyl)methanol

2M Lithium tetrahydroaluminate in tetrahydrofuran (80.0 mL, 160 mmol)was charged into a 500 mL 3-necked round bottomed flask (oven-dried andcooled under argon) fitted with a magnetic stir bar and the solution wascooled to 0° C. under argon. 1-Aminocyclohexanecarboxylic acid (7.64 g,53.3 mmol) is added portionwise over a period of 1 hour. At the end ofthe addition, the reaction mixture was diluted with tetrahydrofuran (60mL), slowly warmed to room temperature, and then heated at reflux for 18hours. The mixture was cooled to room temperature. The reaction mixturewas further diluted with tetrahydrofuran (160 mL) and then cooled to 0°C. Saturated aqueous sodium carbonate (100 ml) was added very slowlykeeping the internal temperature below 15° C. After the addition of thecarbonate solution is complete, the ice bath was left to expire and themixture slowly warmed to room temperature overnight. The reactionmixture was filtered thru a pad of Celite® washing with ethyl acetate(400 mL). The solvent was removed in vacuo to afford a wet oil which wastaken up in methylene chloride (300 mL) and dried over sodium sulfate.Filtration and concentration of the solvent in vacuo affords 6.89 g (99%yield) of the desired product as a clear colorless oil. NMR (CDCl₃) 3.34(s, 2H), 1.81 (bs, 3H), 1.51-1.32 (m, 10H); MS (ESI+) for C₇H₁₅NO m/z130.0 (M+H)⁺.

(1-Aminocyclopentyl)methanol

Using General Procedure L on commercially available cycloleucine affordsthe desired product in 99% yield as a pale yellow oil. NMR (CDCl₃) 3.40(s, 2H), 1.86-1.61 (m, 9H), 1.46-1.29 (m, 2H); MS (ESI+) for C₆H₁₃NO m/z116.1 (M+H)⁺.

General Procedure M.

1-{[(Triisopropylsilyl)oxy]methyl}cyclohexanamine

(1-Aminocyclohexyl)methanol (3.43 g, 26.5 mmol) was taken up inmethylene chloride (80 mL) at room temperature under argon.Triethylamine (5.6 mL, 40 mmol) was added followed by the addition oftriisopropylsilyl chloride (5.34 mL, 25.2 mmol). The reaction mixturewas stirred at room temperature overnight during which time it becameturbid. The reaction mixture was poured into a separatory funneltransferring with methylene chloride (100 mL). The organic layer waswashed sequentially with water (40 mL×2) and brine (40 mL). The organiclayer was dried over sodium sulfate, filtered and the solventconcentrated in vacuo to afford 6.68 g (93% yield) of the desiredproduct as a clear pale yellow oil. NMR (CDCl₃) δ 3.49 (s, 2H),1.75-1.25 (m, 10H), 1.16-1.06 (m, 21H); MS (ESI+) for C₁₁H₂₇NOSi m/z203.2 (M+H)⁺.

1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine

Following General Procedure M and using (1-aminocyclopentyl)methanol thedesired product was obtained in 85% yield as a clear dark yellow oil.NMR (CDCl₃) δ 3.53 (s, 2H), 1.85-1.39 (m, 8H), 1.16-1.07 (m, 21H); MS(ESI+) for C₁₅H₃₃NOSi m/z 272.2 (M+H)⁺.

2-[(Triisopropylsilyl)oxy]ethanamine

Following General Procedure M and using commercially availableethanolamine the desired product was obtained in 99% yield as a clearpale yellow oil. NMR (CDCl₃) δ 3.56 (t, 2H, J=6.0 Hz), 2.94 (t, 2H,J=6.0 Hz), 1.09-0.99 (m, 21H); MS (ESI+) for C₁₁H₂₇NOSi m/z 217.2(M+H)⁺.

1-{[tert-Butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine

Following General Procedure M and using commercially available2-amino-2-methyl-1-propanol and using tert-butyldimethylsilyl chloridethe desired product was obtained in 95% yield as a clear colorless oil.NMR (CDCl₃) δ 3.31 (s, 2H), 0.93 (s, 9H), 0.06 (s, 6H); MS (ESI+) forC₁₀H₂₅NOSi m/z 204.2 (M+H)⁺.

As exemplified in Scheme 10, compounds of Formula VI can be synthesizedbeginning with the aldehyde illustrated above. In Step 1, an alkyne canbe treated with an organic solvent, and a base optionally at a reducedtemperature and subsequently treated with an aldehyde according tomethods known in the art. For example, the aldehyde in Step 1 can betreated with a base, for example, isopropylmagnesium chloride lithiumchloride complex in an organic solvent, for example, tetrahydrofuran atabout −15° C. and next treated with an aldehyde to generate an alkyne.In Step 2, a desired alkynyl alcohol can be treated with a base in anorganic solvent at an elevated temperature to isomerize the desiredalkynyl alcohol to a desired alkene. For example, a desired alkynylalcohol can be treated with a base, such as triethylamine in an organicsolvent, for example, 1,4-dioxane at an elevated temperature of about60° C. to generate an alkene. In Step 3, a desired alkene can be treatedwith ammonia and a mixture of organic solvents to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateaccording to methods known in the art. For example, an alkene can betreated with 0.5M ammonia and a mixture of organic solvents, forexample, dioxane and acetonitrile to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate.In Step 4, a desired ester can be treated with a desired acid and anorganic solvent to generate a desired carboxylic acid according tomethods known in the art. For example, a desired ester can be treatedwith a desired acid, for example, trifluoroacetic acid, to generate acarboxylic acid. In one embodiment, the organic solvent isdichloromethane. In Step 5, a desired acid can be treated with a desiredamine, an organic solvent and a coupling reagent to form a desired amideaccording to methods known in the art. For example, a desired acid canbe treated with a desired amine, an organic solvent, for example,N,N-dimethylformamide, and a coupling reagent, for example,1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, to generate an amide. In Step 6, a silylprotected alcohol can be treated with a fluoride reagent and an organicsolvent according to methods known in the art to generate a desiredalcohol. For example, a silyl protected alcohol can be treated with afluoride reagent, for example, tetrabutylammonium fluoride, and anorganic solvent, for example, acetonitrile, to generate an alcohol. Instep 7, a desired alcohol can be treated with a sulfonyl chloride togenerate a desired mesylate according to methods known in the art. Forexample, an alcohol can be treated with a desired sulfonyl chloride, forexample, methanesulfonyl chloride, to generate a mesylate. In oneembodiment, an amine spontaneously reacts with said mesylate to generatea cyclic amide. In Step 8, a desired thiol can be treated with a desiredamine and an organic solvent at an elevated temperature to generate adesired amine according to methods known in the art. For example, athiol can be treated with an amine, for example,5-(4-methylpiperazin-1-yl)pyridin-2-amine, and an organic solvent, forexample, N,N-dimethylacetamide, at an elevated temperature of about 150°C. to generate an amine. The compound5-(4-methylpiperazin-1-yl)pyridin-2-amine can be prepared as disclosedin U.S. Pat. No. 8,598,186 to Tavares and Strum.

As exemplified in Scheme 11, compounds of Formula VI can be synthesizedbeginning with the aldehyde illustrated above. In Step 1, an alkyne canbe treated with an organic solvent, and a base optionally at a reducedtemperature and subsequently treated with an aldehyde according tomethods known in the art. For example, the aldehyde in Step 1 can betreated with a base, for example, isopropylmagnesium chloride lithiumchloride complex in an organic solvent, for example, tetrahydrofuran atabout −15° C. and next treated with an aldehyde to generate an alkyne.In Step 2, a desired alkynyl alcohol can be treated with a base in anorganic solvent at an elevated temperature to isomerize the desiredalkynyl alcohol to a desired alkene. For example, a desired alkynylalcohol can be treated with a base, such as triethylamine in an organicsolvent, for example, 1,4-dioxane at an elevated temperature of about60° C. to generate an alkene. In Step 3, a desired alkene can be treatedwith ammonia and a mixture of organic solvents to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateaccording to methods known in the art. For example, an alkene can betreated with 0.5M ammonia and a mixture of organic solvents, forexample, dioxane and acetonitrile to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate.In Step 4, an amine can be treated with a base, an organic solvent, anda cyclic sulfamidate to form an amine according to methods known in theart. For example, a desired amine can be treated with a base, forexample, triethylamine, an organic solvent, for example,N,N-dimethylformamide, and a cyclic sufamidate to form an amine. In Step5, a protected amine can be treated with an organic acid to form anamine that can subsequently form a cyclic amide according to methodsknown in the art. For example, a protected amine can be treated with anorganic acid, for example, trifluoroacetic acid, and subsequently reactwith an ester to form a cyclic amide.

EXAMPLES

The patents WO 2013/148748 entitled “Lactam Kinase Inhibitors” toTavares, F. X., WO 2013/163239 entitled “Synthesis of Lactams” toTavares, F. X., and U.S. Pat. No. 8,598,186 entitled “CDK Inhibitors” toTavares, F. X. and Strum, J. C. are incorporated by reference herein intheir entirety.

Example 1 Synthesis of Compound 2 Scheme 1

Compound 2 is synthesized according to the method of A. Haidle et al.,See, WO 2009/152027 entitled“5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives for MARKinhibition.”

Example 2 Synthesis of Compound 3 Scheme 1

Step 1: A round-bottomed flask inerted with a nitrogen atmosphere ischarged with Compound 2, ethanol, and lithium borohydride at ambienttemperature. The reaction is stirred at ambient temperature andmonitored by thin layer chromatography (TLC) or high-performance liquidchromatography (HPLC). Once Compound 2 can no longer be detected, thereaction is quenched with an aqueous acid such as aqueous hydrochloricacid, diluted with ethyl acetate and the layers separated. The organiclayer is dried over anhydrous magnesium sulfate, filtered andconcentrated in vacuo. The product, a primary alcohol, is purified bysilica gel column chromatography eluting with a hexane-ethyl acetategradient and used directly in the next step.

Step 2: A round-bottomed flask inerted with a nitrogen atmosphere ischarged with the primary alcohol prepared in step 1, DMF and phosphorustribromide. The reaction is stirred at ambient temperature and monitoredby thin layer chromatography (TLC) or HPLC. Once the primary alcohol canno longer be detected, the reaction is quenched with brine and dilutedwith toluene. The layers are separated and the toluene layer is driedover anhydrous magnesium sulfate, filtered and concentrated in vacuo.The bromide is purified by silica gel column chromatography eluting witha hexane-ethyl acetate gradient.

Example 3 Synthesis of Compound 5 Scheme 1

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith tetrahydrofuran and the lactam 4, described below. The reaction iscooled to −78° C. and lithium diisopropylamide solution (2M inTHF/heptane/ethyl benzene) is added dropwise. To the resulting enolateis added Compound 3, dropwise, and the reaction is allowed to warm toroom temperature overnight. The reaction is diluted with saturated brineand the layers are separated. The organic layer is dried over anhydrousmagnesium sulfate, filtered and concentrated in vacuo. The product ispurified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Example 4 Synthesis of Compound 6 Scheme 1

A round-bottomed flask is charged with Compound 5 and an aqueous acid,for example a pH=1 HCl solution. The reaction is allowed to stir at roomtemperature until starting material is no longer detected by thin layerchromatography or HPLC. The reaction is neutralized with solid K₂CO₃ anddiluted with dichloromethane. The layers are separated, the organiclayer dried over anhydrous magnesium sulfate, filtered and concentrated.Compound 6 is purified by silica gel column chromatography eluting witha dichloromethane-methanol gradient.

Example 5 Synthesis of Compound 7 Scheme 1

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 6, ethanol and DBU (10 eq). The reaction is monitored bythin layer chromatography or HPLC. Note: The reaction can be heated atreflux if necessary. Once Compound 6 is no longer detected, the reactionis concentrated in vacuo. The lactam 7 is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Example 6 Synthesis of Compound 8 Scheme 1

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 7, meta-chloroperoxybenzoic acid, an organic solvent andstirred at ambient temperature. The reaction is monitored by thin layerchromatography or HPLC. Once Compound 7 is no longer detected, thereaction is concentrated in vacuo. Compound 8 is purified by silica gelcolumn chromatography eluting with a dichloromethane-methanol gradient.

Example 7 Synthesis of Compound 10 Scheme 1

The tricyclic lactam 8 is combined with an amine (9, 0.9 eq) and anorganic solvent such as tetrahydrofuran. A strong base such as lithiumhexamethyldisilazane is added and the reaction is stirred until lactam 8is no longer detected by either thin layer chromatography or HPLC. Thereaction is concentrated in vacuo. The product is purified by silica gelcolumn chromatography eluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thetricyclic lactam 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, andamine 9 (0.9 eq). The reaction is heated at 150° C. for 1-4 hours whilebeing monitored by TLC. Once the tricyclic lactam 8 is no longerdetected by TLC or HPLC, the reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Example 8 Synthesis of Compound 11 Scheme 2

Compound 7 is treated with an oxidizing agent such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in an organic solvent togenerate the alkene intermediate 12.

Example 9 Synthesis of Compound 14 Scheme 2

The sulfone intermediate 12 is combined with an amine (13, 0.9 eq) in anorganic solvent such as tetrahydrofuran. An organic base such as lithiumhexamethyldisilazane is added and the reaction is stirred until sulfoneintermediate 12 can no longer be detected by thin layer chromatographyor HPLC. The product is purified by silica gel column chromatographyeluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thesulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, andamine 13 (0.9 eq). The reaction is heated at 150° C. for 1-4 hours whilebeing monitored by TLC. Once the sulfone intermediate 12 is no longerdetected by TLC or HPLC, the reaction is concentrated in vacuo.

The product is purified by silica gel column chromatography eluting witha dichloromethane-methanol gradient.

Example 10 Synthesis of Compound 4

Step 1: Synthesis of Compound 15 (Scheme 3)

Compound 15 is synthesized according to the method of Arigon, J., See,US 2013/0289031, entitled “Pyrimidinone derivatives, preparation thereofand pharmaceutical use thereof.”

Step 2: Synthesis of Compound 16 (Scheme 3)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 15, dichloromethane and triethylamine (1.5 eq). Thereaction is cooled to 0° C. and Boc anhydride (1.5 eq) is added. Thereaction is allowed to stir at room temperature until Compound 15 is nolonger detected by thin layer chromatography or HPLC. The reaction isconcentrated in vacuo. The product is purified by silica gel columnchromatography eluting with a hexane-ethyl acetate gradient.

Step 3: Synthesis of Compound 17 (Scheme 3)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 16, acetonitrile and a base such as potassium carbonate.Methyl chloroacetate is added dropwise. The reaction is allowed to stirat room temperature until Compound 16 is no longer detected by thinlayer chromatography or HPLC. The reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography eluting with ahexane-ethyl acetate gradient.

Step 4: Synthesis of Compound 18 (Scheme 3)

Compound 17 is dissolved in a solution comprising 3M HCl in methanol andthe reaction is stirred at ambient temperature. Note: the reaction canbe heated at a temperature of about 25° C. to about 60° C. to acceleratethe reaction rate. Once the starting material is no longer detected bythin layer chromatography, the reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography using adichloromethane-methanol gradient

Step 5: Synthesis of Compound 19 (Scheme 3)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 18, dichloromethane, and diisopropylethylamine (1.2 eq).Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwise. Thereaction is allowed to stir at room temperature and monitored by TLC.Once the starting material is no longer detected by thin layerchromatography, the reaction is quenched with saturated brine solution.The organic layer is separated, dried over anhydrous magnesium sulfate,filtered and concentrated in vacuo. The product is purified by silicagel column chromatography using a dichloromethane-methanol gradient.

Step 6: Synthesis of Compound 4

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith anhydrous tetrahydrofuran and Compound 19. The reaction is cooledto −78° C. Sodium bis(trimethylsilyl)amide (1M in THF, 1.1 eq) is addeddropwise. Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwisewith stirring and the reaction is allowed to warm to room temperatureovernight. The reaction is quenched with saturated brine solution andthe layers are separated. The organic layer is dried over anhydrousmagnesium sulfate, filtered and concentrated in vacuo. The product ispurified by silica gel column chromatography using adichloromethane-methanol gradient.

Example 11 Synthesis of Compound 25 Scheme 5

Compound 20 is commercially available. Compound 25 is synthesizedaccording to the synthetic methodology disclosed in Example 10.

Example 12 Synthesis of Compound 31 Scheme 6

Compound 31 is synthesized according to the synthetic methodologydisclosed in Example 10.

Example 13 Synthesis of Compound 33 Scheme 7

Step 1: Synthesis of Compound 32

Compound 32, 5-morpholinopyrid-2-amine, is synthesized according toTavares, F. X. and Strum, J. C., See, U.S. Pat. No. 8,598,186, entitled“CDK Inhibitors”.

Step 2: Synthesis of Compound 33

The sulfone intermediate 8 is diluted with a suitable solvent such astetrahydrofuran and an organic base such as lithium hexamethyldisilazaneis added. The compound 32 is added and the reaction is stirred untilsulfone intermediate 8 can no longer be detected by thin layerchromatography or HPLC. The product is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thesulfone intermediate 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and5-morpholinopyrid-2-amine (0.9 eq). The reaction is heated at 150° C.for 1-4 hours while being monitored by TLC. Once the sulfoneintermediate 8 is no longer detected by TLC or HPLC, the reaction isconcentrated in vacuo. The product is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Example 14 Synthesis of Compound 34 Scheme 8

Step 1: Synthesis of Compound 32

Compound 32, 5-morpholinopyrid-2-amine, is synthesized according toTavares, F. X. and Strum, J. C., See, U.S. Pat. No. 8,598,186, entitled“CDK Inhibitors”.

Step 2: Synthesis of Compound 34

The sulfone intermediate 12 is combined with a suitable solvent such astetrahydrofuran and an organic base such as lithiumhexamethyldisilazane. The amine 32 is added and the reaction is stirreduntil sulfone intermediate 12 can no longer be detected by thin layerchromatography or HPLC. The product is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thesulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and5-morpholinopyrid-2-amine (0.9 eq). The reaction is heated at 150° C.for 1-4 hours while being monitored by TLC. Once the sulfoneintermediate 12 is no longer detected by TLC or HPLC, the reaction isconcentrated in vacuo. The product is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Example 15 Preparation of a Formula V Compound

Step 1: Compound 7 is Boc protected according to the method of A. Sarkaret al. (JOC, 2011, 76, 7132-7140).

Step 2: Boc-protected Compound 7 is treated with 5 mol % NiCl₂(Ph₃)₂,0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, inDMI under CO₂ (1 atm) at 25° C. for 20 hours to convert the methyl thiolderivative into the carboxylic acid.

Step 3: The carboxylic acid from Step 2 is converted to thecorresponding acid chloride using standard conditions.

Step 4: The acid chloride from Step 3 is reacted with N-methylpiperazine to generate the corresponding amide.

Step 5: The amide from Step 4 is deprotected using trifluoroacetic acidin methylene chloride to generate the target compound. The product ispurified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Example 16 CDK4/6 Inhibition In Vitro Assay

Selected compounds disclosed herein were tested in CDK4/cyclinD1,CDK6/CycD3, CDK2/CycA, CDK2/cyclinE, CDK5/p25, CDK5/p35, CDK7/CycH/MAT1,and CDK9/CycT kinase assays by Nanosyn (Santa Clara, Calif.) todetermine their inhibitory effect on these CDKs. The assays wereperformed using microfluidic kinase detection technology (Caliper AssayPlatform). The compounds were tested in 12-point dose-response format insinglicate at Km for ATP. Phosphoacceptor substrate peptideconcentration used was 1.25 μM for all assays (except μM 10 was used forthe CKD7/CycH/MAT1 assay and Staurosporine was used as the referencecompound for all assays. Specifics of each assay are as described below:

CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.2 nM; ATP concentration: 100 μM;Incubation time: 3 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM;Incubation time: 3 hr.

CDK6/CyclinD3: Enzyme concentration: 10 nM; ATP concentration: 300 μM;Incubation time: 3 hr.

CDK5/p25: Enzyme concentration: 0.1 nM; ATP concentration: 20 μM;Incubation time: 3 hr.

CDK5/p35: Enzyme concentration: 0.07 nM; ATP concentration: 20 μM;Incubation time: 3 hr.

CDK7/CycH/MAT1: Enzyme concentration: 5 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK9/CycT: Enzyme concentration: 5 nM; ATP concentration: 10 μM;Incubation time: 17 hr.

TABLE 2 Inhibition of CDK kinases by Tricyclic Lactam Compounds Cdk7/Compound Cdk2/ Cdk2/ Cdk4/ Cdk5/ Cdk5/ Cdk6/ CycH/ Cdk9/ No. CycA CycECycD1 p25 p35 CycD3 MAT1 Cyc T ZZZ * * * * * * * YYY * * *** * * ** *BBBB ** * ** * * ** * * AAAA * * * * * * * * CCCC * * * * * * * *GGGG * * ** * * ** * * * >100 μM ** 10 μM < X > 100 μM *** <10 μM

Example 17 G1 Arrest (Cellular G1 and S-phase) Assay

For determination of cellular fractions in various stages of the cellcycle following various treatments, HS68 cells (human skin fibroblastcell line (Rb-positive)) are stained with propidium iodide stainingsolution and run on Dako Cyan Flow Cytometer. The fraction of cells inG0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle isdetermined using FlowJo 7.2.2 analysis.

Example 18 Cell Cycle Arrest by Tricyclic Lactams in CDK4/6-DependentCells

To test the ability of tricyclic lactams to induce a clean G1-arrest, acell based screening method is used consisting of two CDK4/6-dependentcell lines (tHS68 and WM2664; Rb-positive) and one CDK4/6-independent(A2058; Rb-negative) cell line. Twenty-four hours after plating, eachcell line is treated with a tricyclic lactam compound in a dosedependent manner for 24 hours. At the conclusion of the experiment,cells are harvested, fixed, and stained with propidium iodide (a DNAintercalator), which fluoresces strongly red (emission maximum 637 nm)when excited by 488 nm light. Samples are run on Dako Cyan flowcytometer and >10,000 events were collected for each sample. Data areanalyzed using FlowJo 2.2 software developed by TreeStar, Inc.

Example 19 Inhibition of RB Phosphorylation

The CDK4/6-cyclin D complex is essential for progression from G1 to theS-phase of the DNA cell cycle. This complex phosphorylates theretinoblastoma tumor suppressor protein (Rb). To demonstrate the impactof CDK4/6 inhibition on Rb phosphorylation (pRb), tricyclic lactamcompounds are exposed to three cell lines, two CDK4/6 dependent (tHS68,WM2664; Rb-positive) and one CDK4/6 independent (A2058; Rb-negative).Twenty four hours after seeding, cells are treated with a tricycliclactam compound at 300 nM final concentration for 4, 8, 16, and 24hours. Samples are lysed and protein is assayed by western blotanalysis. Rb phosphorylation is measured at two sites targeted by theCDK4/6-cyclin D complex, Ser780 and Ser807/811 using species specificantibodies.

Example 20 Growth Arrest of Small Cell Lung Cancer (SCLC) Cells

The retinoblastoma (RB) tumor suppressor is a major negative cell cycleregulator that is inactivated in approximately 11% of all human cancers.Functional loss of RB is an obligate event in small cell lung cancer(SCLC) development. In RB competent tumors, activated CDK2/4/6 promoteG1 to S phase traversal by phosphorylating and inactivating RB (andrelated family members). Conversely, cancers with RB deletion orinactivation do not require CDK4/6 activity for cell cycle progression.

Tricyclic lactam compounds are tested for their ability to block cellproliferation in a panel of SCLC cell lines with known genetic loss ofRB. SCLC cells are treated with DMSO or a tricyclic lactam for 24 hours.The effect on proliferation is measured by EdU incorporation. AnRB-intact, CDK4/6-dependent cell line (WM2664 or tHS68) and a panel ofRB-negative SCLC cell lines (H69, H82, H209, H345, NCI417, or SHP-77)are analyzed for growth inhibition by the various tricyclic lactams.

Example 21 Growth Arrest of Rb-Negative Cancer Cells

Cellular proliferation assays are conducted using the followingRb-negative cancer cell lines: H69 (human small cell lungcancer—Rb-negative) cells or A2058 (human metastatic melanomacells—Rb-negative). These cells are seeded in Costar (Tewksbury, Mass.)3093 96 well tissue culture treated white walled, clear bottom plates.Cells are treated with tricyclic lactam compounds at nine point doseresponse dilution series from 10 uM to 1 nM. Cells are exposed tocompounds and then cell viability is determined after either four (H69)or six (A2058) days using the CellTiter-Glo® luminescent cell viabilityassay (CTG; Promega, Madison, Wis., United States of America) followingthe manufacturer's recommendations. Plates are read on a BioTek(Winooski, Vt.) Syngergy2 multi-mode plate reader. The Relative LightUnits (RLU) are plotted as a result of variable molar concentration anddata are analyzed using Graphpad (LaJolla, Calif.) Prism 5 statisticalsoftware to determine the EC₅₀ for each compound.

Example 22 Bone Marrow Proliferation as Evaluated Using EdUIncorporation and Flow Cytometry Analysis

For hematopoietic stem cell and/or hematopoietic progenitor cell (HSPC)proliferation experiments, young adult female FVB/N mice are treatedwith a single dose of the tricyclic lactams described herein by oralgavage. Mice are then sacrificed at 0, 12, 24, 36, or 48 hours followingcompound administration, and bone marrow is harvested, as previouslydescribed (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536).Four hours before the bone marrow is harvested, mice are treated with100 μg of EdU by intraperitoneal injection (Invitrogen). Bone marrowmononuclear cells are harvested and immunophenotyped using previouslydescribed methods and percent EdU positive cells are then determined(Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). In brief,HSPCs are identified by expression of lineage markers (Lin−), Sca1 (S+),and c-Kit (K+).

Example 23 Cellular Wash-Out Experiment

HS68 cells are seeded out at 40,000 cells/well in 60 mm dish on day 1 inDMEM containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycinand 1× Glutamax (Invitrogen) as described (Brookes et al. EMBO J,21(12)2936-2945 (2002) and Ruas et al. Mol Cell Biol, 27(12)4273-4282(2007)). 24 hrs post seeding, cells are treated with a tricyclic lactamcompound or DMSO vehicle alone at 300 nM final concentration of testcompounds. On day 3, one set of treated cell samples are harvested intriplicate (0 Hour sample). Remaining cells are washed two times inPBS-CMF and returned to culture media lacking test compound. Sets ofsamples are harvested in triplicate at 24, 40, and 48 hours.

Alternatively, the same experiment is done using normal Renal ProximalTubule Epithelial Cells (Rb-positive) obtained from American TypeCulture Collection (ATCC, Manassas, Va.). Cells are grown in anincubator at 37° C. in a humidified atmosphere of 5% CO2 in RenalEpithelial Cell Basal Media (ATCC) supplemented with Renal EpithelialCell Growth Kit (ATCC) in 37° C. humidified incubator.

Upon harvesting cells, samples are stained with propidium iodidestaining solution and samples run on Dako Cyan Flow Cytometer. Thefraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phaseDNA cell cycle is determined using FlowJo 7.2.2 analysis.

Example 24 Pharmacokinetic and Pharmacodynamic Properties of TricyclicLactams

Tricyclic lactam compounds described herein can be dosed to mice at 30mg/kg by oral gavage or 10 mg/kg by intravenous injection. Blood samplesare taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing andthe plasma concentrations of the tricyclic lactam compounds aredetermined by HPLC.

Example 25 Metabolic Stability

The metabolic stability of tricyclic lactam compounds can be determinedin human, dog, rat, monkey, and mouse liver microsomes. Human, mouse,and dog liver microsomes are purchased from Xenotech, and Sprague-Dawleyrat liver microsomes are prepared by Absorption Systems. The reactionmixture comprising 0.5 mg/mL of liver microsomes, 100 mM of potassiumphosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM of test compoundis prepared. The test compound is added into the reaction mixture at afinal concentration of 1 uM. An aliquot of the reaction mixture (withoutcofactor) is incubated in shaking water bath at 37° C. for 3 minutes.The control compound, testosterone, is run simultaneously with the testcompound in a separate reaction. The reaction is initiated by theaddition of cofactor (NADPH), and the mixture is then incubated in ashaking water bath at 37° C. Aliquots (100 μL) are withdrawn at 0, 10,20, 30, and 60 minutes for the test compound and 0, 10, 30, and 60minutes for testosterone. Test compound samples are immediately combinedwith 100 μL of ice-cold acetonitrile containing internal standard toterminate the reaction. Testosterone samples are immediately combinedwith 800 μL of ice cold 50/50 acetonitrile/dH2O containing 0.1% formicacid and internal standard to terminate the reaction. The samples areassayed using a validated LC-MS/MS method. Test compound samples areanalyzed using the Orbitrap high resolution mass spectrometer toquantify the disappearance of parent test compound and detect theappearance of metabolites. The peak area response ration (PARR) tointernal standard is compared to the PARR at time 0 to determine thepercent of test compound or positive control remaining at time-point.Half-lives are calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Half-life is calculated basedon t½=0.693k, where k is the elimination rate constant based on theslope plot of natural logarithm percent remaining versus incubationtime.

Example 26 Inhibition of Hematopoietic Stem and/or Progenitor Cell(HSPC) Proliferation

To characterize the effect of tricyclic lactam compound treatment onproliferation of the different mouse hematopoietic cells, 8-week-oldfemale C57Bl/6 mice are given a single dose of vehicle alone (20%Solutol) or a tricyclic lactam compound (150 mg/kg) by oral gavage.Ten-hours later, all mice are given a single i.p. injection of 100 mcgEdU (5-ethynyl-2′-deoxyuridine) to label cells in S-phase of the cellcycle. All treated mice are euthanized 2 hours after EdU injection, bonemarrow cells are harvested and processed for flow cytometric analysis ofEdU-incorporation

Example 27 Inhibition of Differentiated Hematopoietic Cell Proliferation

Using the same experimental protocol as discussed in Example 26 above,the effect of tricyclic lactam compounds on the proliferation ofdifferentiated hematopoietic cells is investigated.

Example 28 Radiomitigation Effects of Tricyclic Lactams

The principal acute toxicities of total body irradiation (TBI) at dosesless than 10 Gy are hematologic manifestations such as granulocytopenia,anemia, thrombocytopenia and lymphopenia. At higher doses of IRexposure, intestinal, cutaneous and neurologic toxicities additionallybecome significant contributors to morbidity and mortality, but thehematologic syndrome has been the principal complication faced byimmediate survivors of a mass casualty radiologic disaster. Tricycliclactams are tested for their ability to protect cells from DNA damageand apoptosis induced by irradiation.

DNA damage is determined using the g-H2A.X assay and apoptosis isdetermined with a Caspase 3/7 assay. For the g-H2AX assay, tHS68 cellsare fixed and stained using the g-H2A.X Phosphorylation Assay Kit (FlowCytometry; Millipore, Temecula, Calif.) by the manufacturer'sinstructions. g-H2AX-positive tHDF cells are then quantified using aCyAn ADP Analyzer (Beckman Coulter, Indianapolis, Ind.) and FlowJoanalysis software (Version 7.2.2; Tree Star, Ashland, Oreg.). For the invitro caspase 3/7 assay, tHDF cells are analyzed directly in the 96-wellplates 24 hours after radiation or staurosporine treatment. Caspase 3/7activation is measured using the Caspase-Glo 3/7 Assay System (Promega,Madison, Wis.) by following the manufacturer's instructions.

For the g-H2AX assay, 30,000 cells are plated per well in 12-wellplates. For the caspase 3/7 assay, 1,000 cells are plated per well in96-well white wall clear bottom plates. Cells are incubated at 37° C. ina humidified atmosphere of 5% CO₂ for 24 hours and then irradiated at 6Gy, 8 Gy, or 10 Gy. Cells are then incubated at 37° C. in a humidifiedatmosphere of 5% CO₂ with 100, 300, or 1,000 nM compound or dimethylsulfoxide (Sigma-Aldrich) vehicle control for an additional 16 hoursprior to analysis.

Example 29 Radiomitigation Effects of Tricyclic Lactams in a Mouse Model

Compounds are tested for mitigation of radiation-induced death in vivoin a mouse model. Wild-type mice, young adult (8-12 weeks of age)C57BL/6 (The Jackson Laboratory) or C3H (Harlan Sprague-Dawley) animalsare used. Animals are irradiated using a 137Cs AECL GammaCell 40Irradiator (Atomic Energy of Canada) or a XRAD320 (Precision XRay Inc.)biological irradiator. Experiments are carried out using the 137Cssource, unless otherwise noted. Mice are dosed at 150 mg/kg compound byoral gavage 12 hours post irradiation for single dose studies. Mice aredosed at 150 mg/kg of compound by oral gavage 12 hours post irradiationand 24 hours post irradiation for two dose studies. Kaplan-Meieranalysis of survival over the next 30 days for both treated and controlgroups are determined.

Example 30 Preparation of Drug Product

The active compounds of the present invention can be prepared forintravenous administration using the following procedure. The excipientshydroxypropyl-beta-cyclodextrin and dextrose can be added to 90% of thebatch volume of USP Sterile Water for Injection or Irrigation withstirring; stir until dissolved. The active compound in the hydrochloridesalt form is added and stirred until it is dissolved. The pH is adjustedwith 1N NaOH to pH 4.3+0.1 and 1N HCl can be used to back titrate ifnecessary. USP sterile water for injection or irrigation can be used tobring the solution to the final batch weight. The pH is next re-checkedto ensure that the pH is pH 4.3+0.1. If the pH is outside of the rangeadd 1N HCl or 1N NaOH as appropriate to bring the pH to 4.3+0.1. Thesolution is next sterile filtered to fill 50 or 100 mL flint glassvials, stopper, and crimped.

This specification has been described with reference to embodiments ofthe invention. The invention has been described with reference toassorted embodiments, which are illustrated by the accompanyingExamples. The invention can, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.Given the teaching herein, one of ordinary skill in the art will be ableto modify the invention for a desired purpose and such variations areconsidered within the scope of the invention.

We claim:
 1. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula I, II, III, IV, or V:

wherein: Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is 2, 3 or 4; each X is independently CH or N; each X′ is independently, CH or N; X″ is independently CH₂, S or NH, arranged such that the moiety is a stable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atoms may optionally combine to form a ring; each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two R¹'s on adjacent ring atoms or on the same ring atom together with the ring atom(s) to which they are attached optionally form a 3-8-membered cycle; y is 0, 1, 2, 3 or 4; R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1 or 2 and n is 0, 1 or 2; R³ and R⁴ at each occurrence are independently: (i) hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring; or R³ and R⁴ together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring; R⁵ and R⁵* at each occurrence is: (i) hydrogen or (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance; R^(x) at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵, -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵, -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵, -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵, -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴, -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵, or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein: said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups may be further independently substituted with one or more -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*, -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*, -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, n is 0, 1 or 2, and m is 0, 1 or 2; R³* and R⁴* at each occurrence are independently: (i) hydrogen or (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance; or R³* and R⁴* together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more R^(x) groups as allowed by valance; and R⁶ is H or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atoms may optionally combine to form a ring; and R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈ cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted or substituted C₁-C₆ alkylamino, unsubstituted or substituted di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted or substituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or (ii) —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³ is R^(A); when compounds comprise a double bond in the 6-membered ring fused to the pyrimidine ring, two R⁸ groups are present and are as defined above; when compounds do not comprise a double bond in the 6-membered ring fused to the pyrimidine ring, four R⁸ groups are present and are as defined above; or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein each R⁸ is independently hydrogen or C₁-C₃ alkyl.
 3. The method of claim 1, wherein the compound has the formula:


4. The method of claim 1, wherein the compound has the formula:


5. The method of claim 1, wherein the compound has the formula:


6. The method of claim 1, wherein the compound has the formula:


7. The method of claim 1, wherein the compound has the formula:


8. The method of claim 1, wherein the compound has the formula:


9. The method of claim 1, wherein the compound has the formula:


10. The method of claim 1, wherein the compound has the formula:


11. The method of claim 1, wherein the compound has the formula:


12. The method of claim 1, wherein the compound has the formula:


13. The method of claim 1, wherein the compound has the formula:


14. The method of claim 1, wherein the compound has the formula:


15. The method of claim 1, wherein the compound has the formula:


16. The method of claim 1, wherein the compound has the formula:


17. The method of claim 1, wherein the compound has the formula:


18. The method of claim 1, wherein the compound has the formula:


19. The method of claim 1, wherein the compound is selected from the group consisting of: Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX


20. The method of claim 1, wherein the subject is a human.
 21. The method of claim 1, wherein the subject's HSPCs return to approximately pre-treatment baseline cell cycle activity prior to the exposure to IR.
 22. The method of claim 1, wherein the compound is administered to the subject prior to the exposure to IR.
 23. The method of claim 1, wherein the subject is undergoing radio-therapy to treat a disease.
 24. The method of claim 1, wherein the subject is being treated for a proliferative disorder.
 25. The method of claim 1, wherein the subject is being treated for a CDK4/6 replication independent cancer.
 26. The method of claim 1, wherein the subject is being treated for an ionizing radiation exposure associated with an environmental or occupational condition.
 27. The method of claim 1, wherein administration of the compound does not affect growth of diseased cells.
 28. The method of claim 1, wherein the subject is further treated with a hematopoietic growth factor upon dissipation of the CDK4/6 inhibitor's inhibitory effect.
 29. The method of claim 28, wherein the hematopoietic growth factor is selected form the group consisting of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steel factor, and erythropoietin (EPO).
 30. The method of claim 1, wherein the compound is administered to the subject prior to exposure to the ionizing radiation, during exposure to the ionizing radiation, after exposure to the ionizing radiation, or a combination thereof.
 31. The method of claim 1, wherein the compound is administered to the subject less than about 24 hours prior to exposure to the ionizing radiation.
 32. The method of claim 1, wherein the compound is administered to the subject prior to exposure to the ionizing radiation such that the compound reaches peak serum levels during exposure to the ionizing radiation.
 33. The method of claim 1, wherein the compound is administered to the subject less than about 4 hours prior to exposure to the ionizing radiation.
 34. The method of claim 1, wherein the compound is administered to the subject after exposure to the ionizing radiation.
 35. The method of claim 1, wherein the compound is administered to the subject about 12 hours or more after exposure to the ionizing radiation.
 36. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as defined in claim 1; each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionally independently substituted with one or more R^(x) groups as allowed by valence, and wherein two R^(x) groups bound to the same or adjacent atoms may optionally combine to form a ring; or two R¹⁴ groups bonded to the same carbon can form an exocyclic double bond; or two R¹⁴ groups bonded to the same carbon can form a carbonyl group; and when the compound of Formula VI has a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, two R¹⁴ groups are present as allowed for in Formula VI above; or when the compound of Formula VI does not include a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, four R¹⁴ groups are present as allowed for in Formula VI above; or a pharmaceutically acceptable salt thereof.
 37. The method of claim 1, wherein the compound is selected from the group consisting of:


38. The method of claim 36, wherein the compound is selected from the group consisting of:


39. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula I, II, III, IV, or V:

wherein: Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is 2, 3 or 4; each X is independently CH or N; each X′ is independently CH or N; X″ is independently CH₂, S or NH, arranged such that the moiety is a stable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valence, and wherein two R^(x) groups bound to the same or adjacent atoms may optionally combine to form a ring; each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two R¹'s on adjacent ring atoms or on the same ring atom together with the ring atom(s) to which they are attached optionally form a 3-8-membered cycle; y is 0, 1, 2, 3 or 4; R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1, or 2 and n is 0, 1 or 2; wherein heterocyclo may be optionally independently substituted with 1 to 3 R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring; R³ and R⁴ at each occurrence are independently: (i) hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring; or R³ and R⁴ together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more R^(x) groups as allowed by valance, and wherein two R^(x) groups bound to the same or adjacent atom may optionally combine to form a ring; R⁵ and R⁵* at each occurrence is: (i) hydrogen or (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valance; R^(x) at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵, -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵, -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵, -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵, -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴, -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—OR⁵, -(alkylene)_(m)-N(R³)—C(S)—OR⁵, or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein: said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups, any of which, other than heterocyclo, may be further independently substituted with one or more -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*, -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*, -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, and wherein heterocycle may be further independently substituted with one to three substitutions selected from -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*, -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*, -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or -(alkylene)_(m)-N(R³*)—SO₂—R⁵*; n is 0, 1 or 2, and m is 0, 1; or 2 and R³* and R⁴* at each occurrence are independently: (i) hydrogen or (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valance; or R³* and R⁴* together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more R^(x) groups as allowed by valance; R⁶ is H, absent, or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valence, and wherein two R^(x) groups bound to the same or adjacent atoms may optionally combine to form a ring; and R¹⁰ is 1 (i) NHR^(A), wherein R^(A) is unsubstituted or substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈ cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted or substituted C₁-C₆ alkylamino, unsubstituted or substituted di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted or substituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or (ii) —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³ is R^(A); when the compound of Formula I, II, III, IV, or V has a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, two R⁸ groups are present as allowed for in Formula I, II, III, IV, or V above; or when the compound of Formula I, II, III, IV, or V does not include a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, four R⁸ groups are present as allowed for in Formula I, II, III, IV, or V above; wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized; wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more R^(x) substituents; wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 R^(x) substituents; or a pharmaceutically acceptable salt thereof.
 40. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as defined in claim 39; each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which, other than heterocyclo, may be optionally independently substituted with one or more R^(x) groups as allowed by valence, and wherein two R^(x) groups bound to the same or adjacent atoms may optionally combine to form a ring; or two R¹⁴ groups bonded to the same carbon can form an exocyclic double bond; or two R¹⁴ groups bonded to the same carbon can form a carbonyl group; and when the compound of Formula VI has a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, two R¹⁴ groups are present as allowed for in Formula VI above; or when the compound of Formula VI does not include a double bond, as indicated by the (----), in the 6-membered ring fused to the pyrimidine ring, four R¹⁴ groups are present as allowed for in Formula VI above; wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized; wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more R^(x) substituents; wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 R^(x) substituents; or a pharmaceutically acceptable salt thereof. 